WO2013064818A1 - Aptamères - Google Patents

Aptamères Download PDF

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Publication number
WO2013064818A1
WO2013064818A1 PCT/GB2012/052702 GB2012052702W WO2013064818A1 WO 2013064818 A1 WO2013064818 A1 WO 2013064818A1 GB 2012052702 W GB2012052702 W GB 2012052702W WO 2013064818 A1 WO2013064818 A1 WO 2013064818A1
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WIPO (PCT)
Prior art keywords
aptamers
aptamer
coli
binding
fluorescence
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PCT/GB2012/052702
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English (en)
Inventor
Riikka KÄRKKÄINEN
Mette Ryun DRASBEK
Niall W. G. YOUNG
Graham A. BONWICK
Christopher Smith
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Dupont Nutrition Biosciences Aps
The University Of Chester
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Application filed by Dupont Nutrition Biosciences Aps, The University Of Chester filed Critical Dupont Nutrition Biosciences Aps
Priority to US14/355,351 priority Critical patent/US20150056627A1/en
Priority to EP12784650.9A priority patent/EP2773759A1/fr
Publication of WO2013064818A1 publication Critical patent/WO2013064818A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to novel nucleic acid aptamers and their uses. BACKGROUND OF THE INVENTION
  • Aptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules such as proteins, bacterial cells, viruses and smaller molecular targets such as organic dyes. They can subsequently be exploited in a fashion analogous to more traditional biomolecules such as antibodies. Aptamers can be chemically synthesised. Therefore, in contrast to antibodies, no ethical issues are involved in aptamer production. The potential of aptamers and the need for development of new aptamers with specificity against pathogenic micro-organisms will be discussed. Food can often be contaminated by a range of pathogenic micro-organisms.
  • Tuerk & Gold and Ellington & Szostak first described specific nucleotide molecules that can be selected to bind to proteins. They called these high-affinity single-stranded DNA or RNA molecules, 'aptamers', a name derived from the Latin term aptus, 'to fit'.
  • aptamers Since their discovery, the techniques for isolating aptamers have been developed (Vivekananda & Kiel, 2003; Hamula et al., 2008; Cao et al., 2009) and aptamers have been targeted to bind to several different targets including proteins, bacterial cells (Hamula et al., 2008), viruses (Symensma et al., 1996), prions (Takemura et al., 2006) and smaller molecular targets such as organic dyes (Ellington & Szostak, 1990).
  • Aptamers can be selected in vitro through the technique described by Tuerk & Gold (1990) as the systematic evolution of ligands by exponential enrichment (SELEX). Once aptamers have been identified they can be inexpensively produced either synthetically or enzymatically (Pendergrast et al., 2005) and no animals or animal derived cells are needed for their production. This has the potential to lead to cheaper production costs when compared to antibodies. Aptamers can also be stored as a lyophilised powder at room temperature for more than one year (Pendergrast et al., 2005) and they can recover their native active conformation after denaturation. They are also more stable at higher temperatures than antibodies, which only normally function under physiological conditions (Tombelli et al., 2007).
  • an object of the present invention is to provide novel aptamers for use in analysing complex matrices, such as food and/or for use in detecting pathogenic microorganisms in a sample (preferably in a complex matrix, such as food).
  • Figure 1 shows the predicted secondary structure for aptamer 6AptK12 - the sequences are given in table 2.1 in Example 1 ;
  • Figure 2 shows FAM-labelled aptamers binding to E. coli K12 cells.
  • Figure 3 shows FAM-labelled aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 (50 pmol) binding to the surface of E. coli K12.
  • Pictures were taken with a fluorescence microscope with 60* magnification with a green (495 nm) light.
  • Figure 4 shows FAM-labelled aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 binding to E. coli K12 extracted from yoghurt.
  • Figure 5 shows FAM-labelled aptamers binding to E. coli 0157 497 cells.
  • FIG 8 shows an illustration of enzyme linked technique for detection of bound aptamers.
  • Biotin labelled aptamer bound to target cell wall and peroxidase (Px) labelled streptavidin (SA) has bound to biotin. The colour change appears when ABTS substrate reacts with peroxidase.
  • Figure 9 shows a schematic of Aptamer cloning. 1. PCR amplification of the aptamer pools. 2. Ligation of the aptamers (insert) into linearised plasmid vector. 3. Transformation of the vector with an aptamer insert into the competent bacterial cells. 4. Growth of bacteria and enrichment of the cloned plasmid during the normal bacterial growth.
  • Figure 10 shows pGEM ® -T Easy Vector map and sequence reference points (Promega Technical manual).
  • Figure 1 1 shows sequence and multi-cloning site of pGEM ® -T Easy Vector (adapted from Promega Technical manual).
  • Figure 12 shows sequence and multi-cloning site of pGEM -T Easy Vector (adapted from Promega Technical manual) with sequencing primer sites (in red).
  • Figure 13 shows 2% Agarose gel with the DNA library and non-specific products. Lane M on the gel contains PCR Sizer 100bp DNA Ladder, lane 0 is a PCR control and in lane 1 is a PCR amplified DNA library with 2.5 pmol template DNA.
  • Figure 14 shows homo- and heterodimers that can be formed between the primers PR1 and PR2.
  • the oligonucleotides were analysed with an Oligoanalyzer.
  • Figure 15 shows 2% Agarose gel with DNA library.
  • Lane M on the gel contains PCR MiniSizer 50 bp DNA Ladder
  • lane 0 is a PCR control sample (no template DNA added)
  • lanes 1 , 2, and 3 are DNA library with 0.1 pmol template DNA
  • lanes 4, 5, and 6 are DNA library with 0.5 pmol template DNA.
  • Figure 16 shows agarose gel (2%) with aptamer pool 1 (a), 2 (b), 3 (c), and 4 (d) with two replicates.
  • Lane M on the gel pictures contain PCR Sizer 100bp DNA Ladder, lane 0 is the PCR control sample. The bacterial control samples are in lane 3 and DNA controls in lane 4.
  • Figure 17 shows agarose gels (2%) with aptamer pool 5 (lanes 1 and 2) before (a) and after counter selection with L bulgaricus (b).
  • Lane M contains PCR Sizer 100bp DNA Ladder and lanes 0 is the PCR control sample. On gel A the bacterial control sample is in lane 3 and DNA control in lane 4. On gel C, template control is in lane 1.
  • Figure 18 shows agarose gel (2%) with aptamer pool 6 (Ap6), and 7 (Ap7).
  • Lane M on gel A contains the PCR Sizer 100bp DNA Ladder and on gel B PCR MiniSizer 50bp DNA Ladder, PCR control samples is in lane 0. The bacterial control samples are in lane 3 and DNA controls in lane 4.
  • On gel B two aptamer pools were produced in replicates (lanes 1.1 Ap7, 1.2Ap7, 2.1 Ap7 and 2.2Ap7).
  • Figure 19 shows agarose gel (2%) with aptamer pool 8 before (a) and after (b) counter selection with B. subtilis and S. typhimurium.
  • Lane M on the gels contains PCR MiniSizer 50bp DNA Ladder and lane 0 shows the PCR control sample (no template DNA added).
  • On gel A aptamer pool 8 is in lane 1.1 , 1.2, 2.1 , and 2.2.
  • gel B aptamer pool 8 after the counter selection is in lanes 1 and 2.
  • Figure 20 polyacrylamide gel (10%) with aptamer pool 9.
  • Lane M on the gel contains PCR MiniSizer 50bp DNA Ladder, lane 0 is PCR control sample.
  • On lane 1 and 2 is aptamer pool 9.
  • the bacterial control sample is on lane 3 and DNA control on lane 4.
  • the PCR was repeated 20 times.
  • Figure 21 shows agarose gel (2%) with biotin-labelled aptamer pool 9.
  • Lane M is PCR MiniSizer 50 bp DNA Ladder
  • lane 0 is a PCR control sample
  • lanes 1-12 are the PCR amplified aptamer biotin-labelled aptamer pool 9.
  • Figure 22 shows agarose gel (2%) with FAM-labelled E. coli K12 binding aptamer pool 9.
  • Lane M on the gel contains PCR MiniSizer 50 bp DNA Ladder, lane 0 is a PCR control sample (no template DNA added) and FAM-labelled aptamers with an aptamer pool 9 are in lanes 1-7.
  • Figure 23 shows FAM-labelled aptamer pool 9 binding to E. coli K12 cells.
  • Figure 24 shows FAM-labelled aptamer pool 9 binding to the surface of E. coli K12. Images were taken with a fluorescence microscope with 60x magnification with a green light (495 nm) and visible light. No aptamers were added to the control sample (0 pmol) while 10 pmol and 50 pmol aptamers were added to the samples.
  • Figure 25 shows FAM-labelled aptamer pool binding to the surface of E. coli K12. Image was taken with a fluorescence microscope with 60* magnification with a green light (495 nm). The long structure circled was a typical finding in fluorescence images that might indicate aptamers binding to the bacterial cells in the division stage of their life cycle.
  • Figure 27 shows optimal binding time of the aptamers.
  • Figure 28 shows fluorescence of non-binding aptamers after the first wash.
  • Figure 29 shows fluorescence of non-binding aptamers after the 2nd and 3rd wash.
  • Figure 30 shows FAM-labelled aptamer pools 3, 5, 7 and 9 binding to E. coli K12 bacterial cells.
  • the fluorescence (495nm, Em 520) measured by the plate reader (n 1 ). Fluorescence values were corrected for background.
  • Figure 31 shows specificity of E. coli K12 specific aptamer pool 9.
  • FAM-labelled aptamers were incubated with E. coli K12 (positive control), E. coli B, B. subtilis and S. aureus.
  • Figure 32 shows specificity of the E. coli K12 binding aptamers. Aptamers were incubated with E. coli K12 (positive control), E. coli B and the images were taken with a fluorescence microscope with 60 ⁇ magnification with a green light (495 nm) and visible light. No aptamers were added to the control sample (0 pmol) while 20 pmol aptamers were added to the samples.
  • Figure 33 shows specificity of the E. coli K12 binding aptamers. Aptamers were incubated with B. subtilis and S. aureus and the images were taken with a fluorescence microscope with 60* magnification with a green light (495 nm) and visible light. No aptamers were added to the control sample (0 pmol) while 20 pmol aptamers were added to the samples.
  • Figure 35 shows dtection of E. coli K12 from a mixture of bacterial cells.
  • FAM-labelled aptamer pool nine was incubated with a mixture of E. coli K12, E. coli B and S. aureus (Mix) and with each strain separately.
  • Figure 36 show specificity of E. coli K12 specific aptamer pool 9.
  • FAM-labelled aptamers were incubated with E. coli K12 (positive control), E. coli B, S. aureus and L acidophil us.
  • Figure 37 shows biotin labelled aptamer pool 9 bound to E. coli K12. FluoSpheres were bound to biotin and the images were taken with a fluorescence microscope with 100* magnification. Biotin labelled aptamers incubated with E. coli K12 (+) and no aptamers added to the negative (-) control sample. Normal sized images are on left hand side column and zoomed images on right hand side. An example where FluoSpheres are binding to bacterial cell is circled.
  • Figure 38 shows FAM-labelled aptamer pool 9 bound to E. coli K12 followed by Live/Dead SacLight straining. The images were taken with a fluorescence microscope with 100* magnification.
  • Figure 39 shows biotin labelled aptamer pool 9 bound to E. coli K12. FluoSpheres were bound to biotin followed by LIVE/DEAD SacLight staining. The images were taken with a fluorescence microscope with 100* magnification. Biotin labelled aptamers incubated with E. coli K12 (+) and no aptamers added to the negative (-) control sample. Green colour indicates the cells are alive whilst red colour indicates the cells are dead.
  • Figure 40 shows agarose gel image of the PCR Spermix HiFi analysis for positive colonies.
  • Lane M on the gel contains PCR MiniSizer 50 bp DNA Ladder and on lane 0 is a PCR control sample.
  • Lane CI1-CI8 are the cloned colonies.
  • Sample c is the plasmid control sample.
  • Figure 41 shows agarose gel image of the restriction (EcoRI) products.
  • Lane M on the gel contains PCR Sizer 100 bp DNA Ladder. Restriction products for cloned plasmids are in lanes CM - CI8.
  • Figure 42 shows agarose gel images for FAM-labelled cloned aptamers CM and CI2.
  • Lane M on the gel contains PCR MiniSizer 50 bp DNA Ladder and lane 0 is a PCR control sample (no template DNA added).
  • Figure 43 shows FA -labe!led cloned aptamers CI1 and CI2 binding to £ coli K12 cells.
  • Figure 44 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25 °C (NaCI 100 mM, MgCI 1 mWI). The two strongest secondary structures for E. coli K12 binding nucleotide sequence (2CI-AptK12). Aptamers 1AptK12 and 2AptK12 has been created by cutting off (*) the possible binding sites from the 100 nt sequence. Isolated sequences are circled. Dots are representing the base-pair interactions.
  • Figure 45 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25 °C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for E. coli K12 binding nucleotide sequence (3CI-AptK12). Aptamers 3AptK12 and 4AptK12 has been created by cutting off (*) the possible binding sites from the 100 nt sequence. Isolated sequences are circled. Dots are representing the base-pair interactions.
  • Figure 46 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25 °C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for £. coli K12 binding nucleotide sequence (4CI-AptK12). Aptamers 5AptK12 and 6AptK 2 have been created by cutting off (*) the possible binding sites from the 100 nt sequence, isolated sequences are circled. Dots represent the base-pair interactions.
  • Figure 48 shows FAM-labelled aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 (50 pmol) binding to the surface of E. coli K12. Images were taken with a fluorescence microscope with 60* magnification with a green (495 nm) and visible light.
  • Figure 49 shows a mixture of FAM-labelled aptamers (1AptK12, 2AptK12, 4AptK12 and 6AptK12) binding to E. coli K12, E. coli B and S. aureus.
  • Figure 51 shows FAM-labelled aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 (20 pmol) binding to E. coli K12, E. coli B and S. aureus.
  • Figure 52 shows FAM-labelled aptamer pool 9 binding to E. coli K12 in tap water.
  • the values are corrected for background.
  • Figure 53 shows FAM-labelled aptamer pool 9 binding to E. coli K12 extracted from yoghurt.
  • the values are presented as means ⁇ s.d.
  • the samples are corrected for background.
  • Figure 54 shows FAM-labelled aptamer pool 9 binding to E. coli K12 extracted from yoghurt.
  • the values are presented as means ⁇ s.d.
  • the vales are corrected for background.
  • Figure 55 shows FAM-labelled aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 binding to E. coli K12 extracted from natural probiotic yoghurt.
  • Figure 56 shows 2% Agarose gel with aptamer pool for E. coli 496 (lanes 1 and 2) and E. coli 0157 497 (lanes 3-4).
  • the bacterial control samples are in lanes 5 and 6 and the DNA control is in lane 7.
  • the M on the gel is a PCR Sizer 100bp DNA Ladder and the 0 is the PCR control.
  • Figure 57 shows aptamer pool 5 (a and b), 6 (c) and 7 (d) for L innocua 17 (black boxes), L monocytogenes 489 (grey boxes) and L. monocytogenes 490 (white boxes) on 2% agarose gel.
  • the bacterial control samples are in lanes 7, 8 and 9 (c) and in lane 7 (d).
  • the DNA control is in lane 10 (c) and 8 (d).
  • the M on the gel is a PCR Sizer 100bp DNA Ladder and the 0 is the PCR control.
  • Figure 58 shows aptamer pool 8 (a) and 9 (b) for L. monocytogenes 490 (white boxes) on 2% agarose gel.
  • Lane M on the gel is a PCR Sizer 100bp DNA Ladder and lane 0 is the PCR control.
  • Bacterial control is in lane 3 and the DNA control in lane 4 (a).
  • Figure 59 shows aptamer pools 1 (a), 2 (b), 3 (c) and 4 (d) for S. typhimunum 223 (black boxes) and S. enteritidis 1152 (white boxes) on agarose gel (2%).
  • Lane M is a PCR Sizer 100bp DNA Ladder and lane 0 is the PCR control.
  • Bacterial control sample is in lane 3 for 223 and in lane 6 for 1152.
  • the DNA control sample is in lane 7.
  • Figure 60 shows S. typhimurium 223 (black box) and S. enteritidis 1 152 (white box) aptamer pool 5 on agarose gel (2%).
  • the M on the gel is a PCR Sizer 100bp DNA Ladder and in lane 0 is the PCR control.
  • Bacterial control samples are in lanes 3 and 6 and the DNA control sample is in lane 7.
  • Figure 61 shows aptamer pools 6 (a), 7 (b), 8 (c) and 9 (d) for S. typhimurium 223 (black boxes) and S. enteritidis 1152 (white boxes) on agarose gel (2%).
  • Lane M on the gels is a PCR Sizer 100bp DNA Ladder and in lane 0 is the PCR control.
  • Bacterial control samples are in lanes 1 and 2 on gel A and in lane 5 on gel C.
  • the DNA control samples are in lane 3 (a) and in lane 6 on gel c.
  • Figure 62 shows 2 % Agarose gel with the PCR Spermix HiFi analysis for positive colonies.
  • Lane M on the gel contains PCR MiniSizer 50 bp DNA Ladder.
  • CI1 s-CI6s are the cloned colonies for S. typhimurium aptamers and in lanes CI1 e-CI6e are the cloned colonies for E. coli aptamers.
  • lane 0 is a PCR control sample and N is negative control.
  • Figure 63 shows 2% Agarose gel with the purified plasmid vectors.
  • Lane M on the gel contains FullRanger 100 bp DNA Ladder.
  • CI1s-CI6s are the positive clones for S. typhimurium aptamers and in lanes CI2e-CI6e are the positive clones for E. coli aptamers.
  • Figure 64 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25°C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for E. coli 0157 497 binding nucleotide sequence (3CI-Apt497). Aptamer 1Apt497 has been created by cutting off (*) the possible binding sites from the 100 b sequence. Isolated sequences are circled. Blue and red dots are representing the base-pair interactions.
  • Figure 65 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25 °C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for E. coli 0157 497 binding nucleotide sequence (4CI-Apt497). Aptamers 2Apt497 and 3Apt223 has been created by cutting off (*) the possible binding sites from the 100 b sequence. Isolated sequences are circled. Blue and red dots represent the base-pair interactions.
  • Figure 66 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25°C (NaCI 100 mM, MgCI 1 mM).
  • Figure 67 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25°C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for S. typhimurium 223 binding nucleotide sequence (1 CI-Apt223). Aptamers 1Apt223 and 2Apt223 has been created by cutting off ( * ) the possible binding sites from the 100 b sequence. Isolated sequences are circled. Blue and red dots represent the base-pair interactions.
  • Figure 68 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25°C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for S. typhimurium 223 binding nucleotide sequence (2CI-Apt223). Aptamer 3Apt223 has been created by cutting off (*) the possible binding site from the 100 b sequence. Isolated sequences are circled. Blue and red dots represent the base-pair interactions.
  • Figure 69 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25°C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for S.
  • typhimurium 223 binding nucleotide sequence (3CI-Apt223).
  • Aptamers 4Apt223 and 5Apt223 has been created by cutting off (*) the possible binding sites from the 100 b sequence. Isolated sequences are circled. Blue and red dots represent the base-pair interactions.
  • Figure 70 shows predicted aptamer secondary structures (OligoAnalyzer 3.1 , UNAFold) at 25°C (NaCI 100 mM, MgCI 1 mM). The two strongest secondary structures for S. typhimurium 223 binding nucleotide sequence (4CI-Apt223). Aptamer 6Apt223 has been created by cutting off (*) the possible binding sites from the 100 b sequence. Isolated sequences are circled. Blue and red dots represent the base-pair interactions.
  • Figure 71 shows FAM-labelled aptamers binding to E. coli 0157 497 cells.
  • Figure 72 shows microscopy images of FAM-labelled aptamers 1Apt497, 2Apt497 and 4AptK12 (20 pmol) binding to the surface of E. coli 0157 497. Images were taken with a fluorescence microscope with 100* magnification with a green (495 nm) and visible light.
  • Figure 74 shows microscopy images of FAM-labelled E. coli 0157 aptamers 1Apt497, 2Apt497 and 4Apt497 (20 pmol) binding to the surface of E. coli K12. Images were taken with a fluorescence microscope with 60* magnification with a green (495 nm) and visible light.
  • Figure 76 shows microscopy images of FAM-labelled aptamers 2Apt223, 3Apt223 and 5Apt223 (20 pmol) binding to the surface of S. typhimurium 223. Images were taken with a fluorescence microscope with 100 ⁇ magnification with a green (495 nm) and visible light.
  • Figure 78 shows microscopy images showing the binding of the FAM-labelled aptamer 3Apt223 to S. typhimurium and S. enteritidis. Images were taken with a fluorescence microscope with 100* magnification with a green (495 nm) and visible light.
  • Figure 79 shows microscopy images showing the binding of the FAM-labelled aptamer 3Apt223 to E. coli K12 and L plantarum. Images were taken with a fluorescence microscope with 100x magnification with a green (495 nm) and visible light.
  • a seminal finding of the present invention is the development of novel aptamers and the fact that these aptamers have high specificity for pathogenic microorganisms (particularly pathogenic bacteria).
  • the inventors have shown that the aptamers of the present invention have specificity for live pathogenic microorganisms (particularly pathogenic bacteria).
  • the inventors have demonstrated the feasibility of using the aptamers of the present invention in complex matrices (e.g. real food systems) to target and detect specific microorganisms (e.g. microbial food contaminants).
  • the inventors have developed the aptamers of the present invention using a novel selection method using centrifugation (see section 2.3.6 below).
  • the present invention provides a nucleic acid aptamer which specifically binds a pathogenic microorganism, preferably a pathogenic bacterium.
  • a nucleic acid aptamer comprising the nucleotide sequence shown herein as SEQ ID No. 5, 6, 7, 8, 9, 10, 1 , 2, 3 or 4, or a fragment thereof, or a sequence which is at least 80% identical therewith, or a sequence which hybridises under stringent conditions therewith.
  • the present invention provides a kit comprising at least one nucleic acid aptamer according to any one of the preceding claims together with instructions on how to use the at least one nucleic acid aptamer.
  • the present invention yet further provides a device (preferably a hand-held or portable device) comprising at least one of the nucleic aptamers of the present invention or capable of detecting at least one of the nucleic aptamers of the present invention.
  • a device preferably a hand-held or portable device
  • the present invention further provides a microarray or biosensor comprising at least one of the nucleic acid aptamers of the present invention.
  • a further aspect of the present invention provides a method of detecting a microorganism in a sample comprising admixing a nucleic acid aptamer according to the present invention with the sample and identifying the presence of a bound aptamer.
  • a nucleic acid aptamer according to the present invention for detecting a microorganism in a sample.
  • a method of selecting aptamers wherein the aptamer is selected on its ability to bind (e.g. specifically bind) to live bacterial cells, preferably live pathogenic bacterial cells
  • which method comprises the steps of exposing an aptamer to live bacterial cells (preferably live pathogenic bacterial cells) and selecting an aptamer which binds (e.g. specifically) binds to said live bacterial cells, optionally said method further comprises a washing and centrifuging (e.g. at 3500-4000 g for 5min at 4°C) step.
  • said method of selecting aptamers comprises two washing and centrifuging (e.g. at 3500-4000 g for 5min at 4°C) steps. When the method comprises two washing and centrifuging steps one occurs before aptamer binding and the other one occurs after aptamer binding.
  • the nucleic acid aptamer according to the present invention comprises a nucleotide sequence shown herein as SEQ ID No. 5, 6, 7, 8, 9 or 10, or a fragment thereof, or a sequence that hybridises under stringent conditions thereto.
  • nucleic acid aptamer according to the present invention comprises at least 10, preferably at least 20, preferably at least 30, more preferably at least 40 nucleotides.
  • nucleic acid aptamer according to the present invention comprises at most 70 nucleotides in length, preferably at most about 60 nucleotides in length.
  • nucleic acid aptamer according to the present invention comprises in the region of 40 to 60 nucleotides, preferably 42-59 nucleotides. In one embodiment the nucleic acid aptamer according to the present invention comprises about 50 nucleotides.
  • the nucleic acid aptamer has specificity against a live pathogenic bacterium.
  • specificity means that the aptamer is selectively reactive with live pathogenic bacteria compared with either dead pathogenic bacteria or live non-pathogenic bacteria.
  • aptamers have specificity for a particular genera, species or strain of pathogenic bacteria.
  • the aptamer may be selective for Salmonella spp. (such as Salmonella typhimurium, e.g. Salmonella typhimurium 233, Salmonella enteritidis), Escherichia coli spp. (such as E. coli 0157) or Listeria spp.
  • Salmonella spp. such as Salmonella typhimurium, e.g. Salmonella typhimurium 233, Salmonella enteritidis
  • Escherichia coli spp. such as E. coli 0157
  • Listeria spp Listeria spp.
  • specificity would mean that the aptamer preferentially selects that genera, species or strain over any other genera, species or strain and/or that there is no or insignificant cross- reactivity with other genera, species or strains.
  • nucleic acid aptamers are synthetic.
  • the aptamers according to the present invention may be used in a fashion analogous to antibodies. Like antibodies the aptamers provide target binding specificity.
  • the aptamers may be modified by addition of one or more reporter labels (or detectable labels).
  • the label may be attached to either the 5' or 3' end of the aptamer. In a preferred embodiment the label may be attached to the 5'-end of the aptamer.
  • the aptamer may be synthesized by Eurofins MWG Operon, Modified DNA oligos (Oligos a la carte) and FAM (6-carboxyfluroescein) may be attached to the 5'- end.
  • the nucleic acid aptamer comprise a detectable label.
  • the detectable label may be attached directly or indirectly to the nucleic acid aptamer. If the label is indirectly attached to the nucleic acid aptamer this may be by any mechanism known to one of skill in the art, such as using biotin and streptavidin.
  • the aptamer may comprise a reporter label, such as a fluorescent dye or an enzyme.
  • the aptamer may comprise a fluorescent label.
  • the reporter label may comprise one or more parameter(s) for detection.
  • the parameters may be for example the size of the label and/or the optical properties of the label
  • the optical properties are selected from the group consisting of: light reflectivity, colour, the fluorescence emission wavelength(s) and the fluorescence emission intensity.
  • the properties of each label may be measured using microscopy.
  • the microscopy method is selected from the group consisting of bright field microscopy, phase-contrast microscopy, oblique illumination microscopy, dark field microscopy, differential interference contrast microscopy, reflection contrast microscopy, contrast microscopy, polarizing microscopy, interference microscopy and fluorescence microscopy.
  • UV illumination may be used to detect labelled (e.g. fluorescently labelled) aptamers.
  • the detected aptamers may be bound direct to a target (e.g. a microorganism, such as a pathogenic microorganism).
  • the fluorophore is selected from the group consisting of a fluorophore that emits a blue, green, near red or far red fluorescence.
  • the fluorophores do not quench each other.
  • the sizes are selected from the group consisting of about 1 ,9 ⁇ , about 4.4 ⁇ , about 5.4 ⁇ , about 5.8 ⁇ , about 7.4 ⁇ , about 9.7 ⁇ and about 9.8 ⁇
  • the fluorophore is selected from the group consisting of UV2, Starfire Red and TRITC.
  • the aptamer may comprise biotin (or be modified to include biotin) for binding with streptavidin.
  • the aptamer may be pegylated, for example to minimise degradation if used therapeutically in vivo.
  • the aptamer(s) of the present invention may be immobilised on (e.g. bound or adhered to) a substrate or carrier, e.g. a microcarrier.
  • the aptamer(s) of the present invention may be immobilised on a magnetic bead, or micro bead.
  • the microcarrier is a porous or a solid microcarrier.
  • the porous microcarrier is selected from the group consisting of Cytopore microcarrier (e.g. a Cytopore 1 microcarrier or a Cytopore 2 microcarrier), a Cultispher microcarrier, a Cultispher-G microcarrier, a Cultispher-GL microcarrier and a Cultispher-S microcarrier, an Informatrix microcarrier, a Microsphere microcarrier, a Siran microcarrier, and a Microporous MC microcarrier.
  • Cytopore microcarrier e.g. a Cytopore 1 microcarrier or a Cytopore 2 microcarrier
  • Cultispher microcarrier e.g. a Cytopore 1 microcarrier or a Cytopore 2 microcarrier
  • Cultispher microcarrier e.g. a Cytopore 1 microcarrier or a Cytopore 2 microcarrier
  • Cultispher microcarrier e.g
  • the solid microcarrier is selected from the group consisting of a Cytodex microcarrier (eg. a Cytodex 1 , Cytodex 2 or Cytodex 3 microcarrier) a Biosilon microcarrier, a Bioglass microcarrier, a FACT III microcarrier or a DE 52/53 microcarrier.
  • a Cytodex microcarrier eg. a Cytodex 1 , Cytodex 2 or Cytodex 3 microcarrier
  • Biosilon microcarrier eg. a Cytodex 1 , Cytodex 2 or Cytodex 3 microcarrier
  • Bioglass microcarrier e.g. a Bioglass microcarrier
  • FACT III microcarrier e.g. a FACT III microcarrier
  • the aptamer(s) may be used in a device, a microarray, a biosensor, a rapid detection test such as a lateral flow assay (dipstick) or a microplate based assay (e.g. analogous to ELISA).
  • a rapid detection test such as a lateral flow assay (dipstick) or a microplate based assay (e.g. analogous to ELISA).
  • the present invention further provides a microarray or biosensor comprising at least one of the nucleic acid aptamers of the present invention.
  • the microarray or biosensor may comprise more than one aptamer (optionally in combination with one or more antibodies) wherein at least one of the aptamers is an aptamer in accordance with the present invention.
  • the device in accordance with the present invention may be a lateral flow device.
  • a lateral flow assay may also be known as a Lateral Flow
  • a lateral flow device is intended to detect the presence (or absence) of a target analyte in a sample (e.g. complex matrix). Most commonly these tests are used for medical diagnostics either for home testing, point of care testing or laboratory use.
  • the later flow device may be in a dipstick format.
  • a lateral flow test is a form of assay in which the test sample flows along a solid substrate via capilliary action. After the sample is applied to the test it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with the aptamer. Depending upon the analytes present in the sample the coloured reagent can become bound at the test line or zone. Thus the lateral flow device may give rise to a coloured band or spot.
  • the device in accordance with the present invention may be a microplate.
  • the term microplate as used herein may also be referred to as a microtitre plate or microwell plate.
  • the microplate may be a flat plate with multiple "wells" used as small test tubes.
  • the microplate may have 6, 12, 24, 48, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix.
  • the microrarray or biosensor may comprise at least one nucleic acid aptamers of the present invention bound to a microcarrier.
  • the aptamer(s) of the present invention may be used in combination with an antibody (e.g. a target specific antibody) to produce a hybrid assay.
  • an excitation light generating source including but not limited to a light emitting diode, a tungsten light, a halogen light or laser;
  • a means to detect the emitted signal including but not limited to a photo diode or a photomultiplier tube.
  • the aptamers may be used in the selective purification and/or extraction of target molecules (e.g. microorganisms) from mixtures. This can be useful in pre- concentration steps.
  • the aptamer(s) may be immobilised on a carrier (such as magnetic beads).
  • the nucleic acid aptamer according to the present invention may be supplied in a kit.
  • the kit according to the present invention may be a rapid detection test kit.
  • the kit may for example comprise i) at least one (such as 2, 3 or 4) labelled nucleic acid aptamer(s) according to the present invention and ii) instructions on how to use the aptamer(s).
  • the kit may comprise i) at least one fluorescently labelled nucleic acid aptamer according the present invention.
  • the kit of the present invention may further comprise a microcarrier.
  • the microcarrier in the kit may be in a separate container to the nucleic acid aptamer(s).
  • the kit may comprise nucleic acid aptamer(s) bound or adhered to a carrier, e.g. microcarrier.
  • the kit of the present invention may further comprise one or more antibodies, e.g. a target specific antibody.
  • the kit may comprise i) a biotin labelled aptamer and ii) an enzyme labelled streptavidin which enzyme reacts to provide a detectable label.
  • the enzyme may be a peroxidase.
  • the kit may optionally comprise iii) a 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid (ABTS) substrate and/or iv) hydrogen peroxide.
  • ABTS 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid
  • the kit according to the present invention comprises more than one (e.g. at least 2, such as at least 3) nucleic acid aptamers. In one embodiment the kit according to the present invention may comprise:
  • a fluorescently labeled aptamer specific to a target of interest e.g. a microorganism
  • a target of interest e.g. a microorganism
  • a binding reagent to enable binding of the aptamer to said target
  • the aptamers and/or kit may be used in a detection method (or detection assay) for detecting the presence of a microorganism in a sample.
  • the microorganism is a bacterium.
  • the microorganism is a pathogenic bacterium.
  • the pathogenic microorganism is selected from the group consisting of Salmonella spp. (such as Salmonella typhimurium, Salmonella enteritidis), Escherichia coli spp. (such as E. coli 0157) or Listeria spp.
  • Salmonella spp. such as Salmonella typhimurium, Salmonella enteritidis
  • Escherichia coli spp. such as E. coli 0157
  • Listeria spp Listeria spp.
  • the aptamers of the present invention may be used to detect coliform bacteria in a food or water sample.
  • Coliform bacteria are usually present in in large numbers in the faeces of warm-blooded animals, and their detection in water and/or food samples can indicate contamination of the water or food. Coliform bacteria themselves may not cause serious illness, however their presence is used to indicate that other pathogenic organism of faecal original may be present. Therefore in one embodiment of the present invention the microorganism detected by aptamers may be a coliform bacterium, which may or may not be a pathogenic microorganism. Typical genera of coliform bacteria include Citrobacter, Enterobacter, Hafnia, Klebsiella, Serratia, Escherichia.
  • the sample is a complex matrix.
  • the aptamers according to the present invention may be used in a diverse range of diagnostic methods.
  • the sample (which is preferably a complex matrix) may be a food or feed sample, a beverage, a pharmaceutical sample, or a personal care sample.
  • the sample (which is preferably a complex matrix) may be a raw ingredient, a finished product or may be taken from the environment of manufacture or storage.
  • complex matrix we mean a sample which comprises more than one component.
  • the complex matrix may be a food or feed product, a beverage, a pharmaceutical product, or a personal care product.
  • the complex matrix is a food or feed - it may be meat or a meat product (e.g. raw or cooked meat product).
  • admixing means bringing the nucleic acid aptamer according to the present invention into contact with the sample. This may include bringing the aptamer into contact with the surface of a sample, for example the surface of meat or a meat product.
  • the complex matrix is a food or feed - it may be a dairy product or a composition used in the production of a dairy product, such as cheese or yoghurt.
  • the complex matrix is a food or feed - it may be a vegetable based food product.
  • the complex matrix is a food or feed - it may be a ready to eat food or a food ingredient.
  • the complex matrix is a food or feed - it may be a salad product, such as packaged vegetables, e.g. packaged lettuces.
  • the complex matrix is a food or feed - it may be infant formula.
  • the present invention may be used to detect for contamination of a food or feed with spoilage microorganism (e.g. spoilage bacteria) and/or pathogenic microorganisms (e.g. pathogenic bacteria).
  • the sample may be a personal care product such as an eye care product, such as contact lens solution.
  • the sample may be a beverage, such as beer or a sample taken during the brewing of beer. In other words the present invention may be used to detect beer spoilage bacteria.
  • the aptamers according to the present invention may be used in a manufacturing plant to detect for the presence of spoilage bacteria on or in equipment used therein.
  • the aptamers of the present invention may be find use in public health applications.
  • the aptamers may be used to detect the presence of pathogenic bacteria in drinking water for instance.
  • the term "beverage” as used herein includes drinking water.
  • the aptamers of the present invention may be used to detect faecal contamination of drinking water.
  • any unbound aptamer may be washed off before detecting the presence of bound aptamer.
  • the method of the present invention may comprise a further step of washing the admixture of aptamer and sample in order to remove any unbound aptamer.
  • the method of detecting the microorganism in a sample may comprise the steps of admixing the at least one aptamer with the sample, optionally centrifuging the sample and optionally washing the sample, followed by detecting the bound aptamer(s).
  • the aptamer may be admixed with the sample for at least 45 minutes before detecting the presence of bound aptamer.
  • the sample and aptamer are admixed for about 1 hour before detection of the presence of bound aptamer.
  • aptamer means a single stranded DNA or RNA molecule, e.g. a high-affinity single stranded DNA or RNA molecule.
  • high-affinity means that the aptamer readily (and preferably selectively) combines with the microorganism of interest.
  • the aptamers of the present invention may be used for direct detection of a microorganism in a sample.
  • the aptamers of the present invention may be used for direct detection of a microorganism in a complex matrix.
  • the method according to the present invention is an in vitro method.
  • live means that the microorganism, preferably bacterium, is capable of actively dividing or is actively dividing.
  • the aptamers of the present invention may be used for direct detection of a pathogenic bacterium in a complex food matrix.
  • pathogenic means harmful to human or animal health.
  • the pathogenic microorganism, e.g. pathogenic bacteria, may be one which causes food poisoning in humans.
  • aptamer(s) used in accordance with the present invention may be determined by one of ordinary skill in the art. In any event, by way of guidance only it is envisaged that approximately 20 to lOOpmol of aptamer per 100 pm of sample would be sufficient for detection of the target microorganism(s). In one preferred embodiment 50pmol of aptamer/ 100 pm of sample may be used.
  • Aptamers can be used in a fashion analogous to antibodies because they exhibit target binding specificity. However aptamer production does not have moral and ethical issues associated with antibody production.
  • One advantage of the present invention is that the method of detecting a microorganism in a sample in accordance with the present invention does not require enrichment of the microorganism (e.g. culturing of the microorganism) before detection can be carried out. This means the method is easy and fast. As the aptamers are specific for the pathogenic or spoilage microorganism being tested - false positives can be kept to a minimum.
  • aptamers can be stored as a lyophilised powder at room temperature for more than one year. In addition aptamers can recover their native active conformation after denaturation.
  • Biosensors are devices for the detection of biological analytes. Biosensor applications can differentiate biological recognition elements such as enzymes, antibodies and nucleic acids, to detect the target molecule.
  • Aa typical biosensor contains three components: a biological sensing element that can recognise or bind the analyte, a transducing element which converts the detection event into a measurable signal, and a display that transforms the signal into a digital format.
  • the sensing element primarily defines the selectivity and sensitivity of the biosensor.
  • the detection of the analytes is usually based on sensing the analytes with either an electrical (Liss et al., (2002), Analytical Chemistry, 74, 4488-4495; Tombelli et al average 2005 Biosensors and Bioelectronics, 20, 2424-2434; Liu et al, 2009 Electrochimica Acta, 54, 6207-6211 ) or optical (Baldrich et al., 2004 Analytical Chemistry, 76, 7053-7063; Wang et al., 2007b Analytical and Bioanalytical Chemistry, 389, 819-825; Lautner et al., 2010 The Analyst, 135, 918-9; Ohk et al., 2010 Journal of Applied Microbiology, 109, 808-817) readout, each of these references in incorporated herein by reference.
  • a problem in development of biosensors is the failure of most biomolecules to produce an easily measured signal upon target binding. For example, antibodies normally do not change their shape or dynamics when
  • Biosensor technology is currently creating interest because it promises equally reliable results in a shorter time compared to more traditional detection methods such as PCR, colony count, and ELISA.
  • Some examples of rapid biosensor platforms for the detection of bacteria will be now introduced.
  • a highly sensitive and specific RNA biosensor for the rapid detection of viable E. coli in water was developed by Baeumner et al. (2003) Biosensors and Bioelectronics, 18, 405-413. This biosensor can detect as few as 40 bacterial cells in 15-20 minutes. The detection of this portable, inexpensive and very easy to use biosensor was based on the amplification of mRNA.
  • a biosensor to detect food-borne pathogens was developed by Muhammed- Tahir & Alocilja (2003) Biosensors and Bioelectronics, 18, 813-819. It was a conductometric biosensor that provided a specific, sensitive, low volume, and near real-time detection mechanism for food-borne pathogens.
  • the biosensor is based on electrochemical immunoassay which are biosensors constructed with antibodies as biological elements, attached to an electrochemical transducer. In their study the enterohemorrhagic E. coli 0157:H7 and Salmonella ssp. which are of concern to biosecurity were used. It was suggested that the method can be changed for detection of other food-borne pathogens by changing the specificity of the antibodies.
  • antibodies used as biological elements may be replaced with aptamers in biosensor applications. This change enables a rapid method to detect pathogenic bacteria.
  • the advantages of using aptamers over antibodies include the lower costs of production and there are no ethical issues when aptamers are used because they can be produced by a chemical synthesis where no animals or animal cells are needed.
  • Aptamer-based biosensors can be used for the detection of pathogenic microorganisms and viruses.
  • aptamers can be a very good substitute for antibodies because they are easy to handle and they are stable compared to biologically generated proteins.
  • Aptasensors also provide an advantage in chemical stability compared to antibody based affinity biosensors (Liu et al., 2010 Electrochimica Acta, 54, 6207-6211 ).
  • the device and/or biosensor according to the present invention may comprise elements from biosensors and/or aptasensors as disclosed herein.
  • the aptamers according to the present invention may be used in any known biosensor and/or aptasensor.
  • Aptamers could be used as biological recognition elements of biosensors. That an aptamer has bound to its target does not mean it can be used in a biosensor as it is necessary to have a measurable signal from a binding event between the aptamer and the target. When the aptamers bind to their target they usually undergo significant conformational changes. This has been suggested to be one of the key factors when designing the aptamer based biosensors (Wang ei a/. Analytical and Bioanalytical Chemistry, 389, 819-825 2007b; Zhang et ai., 2008 Small, 4, 1 196-1200). Aptasensors have created interest because they are easy to handle and they are stable compared to biologically generated proteins. They are also chemically more stable than antibody-affinity biosensors.
  • the aptamer based biosensors often use immobilised aptamers as recognition elements for the target molecules.
  • the most popularly used electrode material is gold where the thiolated DNA/RNA strands, in this case aptamers, can be immobilised via strong Au-S linkage (Heme ei a/., 1997 Journal of American Chemistry Society, 119, 8916-8920; Steel ei a/., 1998 Analytical Chemistry, 70, 4670-4677).
  • a streptavidin-biotin linkage has also been used (Hamula et ai, 2008 Trends in Analytical Chemistry, 25, 681-691 ;Joshi et ai, 2009
  • RNA and DNA aptamers have been used in biosensors. It has been established that the unmodified RNA aptamer based biosensors can be used only for a single measurement in biological media because of the degradation of the RNA by the ribonucleases (McCauley ei a/., 2003 Analytical Biochemsitry, 319, 244-250.). DNA aptamer based assays have been shown to be reusable with minimal or no change in sensitivity (Lee & Walt, 2000 Analytical Biochemistry, 282, 142-146; Liss et al.
  • aptamers according to the present invention may be used any of known biosensor or aptasensor.
  • the aptamers of the present invention may be used with one or more of these biosensors.
  • biosensor as used herein may be any one of the biosensors or aptasensors taught herein and which comprises the aptamers of the present invention.
  • the aptamer biosensors are roughly divided into two different groups: the biosensors where the interaction between the aptamer and the analyte is detected by optical readout, or by electrochemical readout.
  • Optical platforms use colour or fluorescence labels in detection of the aptamer binding to the analyte.
  • Biosensors based on surface plasmon resonance (SPR) can be used for label-free analysis of biomolecular interactions, providing data on selectivity, affinity and kinetics (Naslund ei a/., 2006 Nature Methods Application Notes, 14-16). SPR has been used to study the interactions between the aptamers and their targets (Baldrich et al.
  • Fluorescence signalling aptamers have been used in many aptasensors and different fluorescence based techniques have been extensively reviewed by Nutiu & Li (2005) Methods, 37, 16-25. The same research group developed a structure-switching signalling aptamers (Nutiu & Li, 2003; 2004 (supra)). Their non-fluorescent aptamer can be turned into fluorescencesignalling reporter when the target molecule is available. In their study the aptamer binds to a DNA sequence modified with a quencher (QDNA) in the absence of the aptamer target. When the system is exposed to the target analyte the aptamer binds to the target instead of the QDNA.
  • QDNA quencher
  • aptamer beacons Similar to structure-switching aptasensors, aptamer beacons (Hall et al., 2009, supra) that work in similar way to the molecular beacons (Tyagi & Kramer, 1996 Nature Biotechnology, 14, 303-308) has been reported. An interaction of the aptamer beacons with the analytes leads to a separation of fluorophore and quencher. Hall et al. (2009) (supra) generated a series of thrombin aptamer beacons. A fluorophore and quencher were included in the aptamer. An addition of thrombin leads to a conformation change of the aptamer and a separation of the fluorophore from the quencher. They developed two different
  • thrombin aptamer beacons that had fast activation rates at 25 oC.An aptamer array sensor was developed for the multiplex detection of four analytes in biological matrix such as human serum or cellular extract (McCauley et al., 2003 (supra)).
  • the optical colorimetric signalling has been reported extensively by Liu & Lu (2006) (supra). They developed a fast colorimetric sensor based on the disassembly of nanoparticle aggregates linked by aptamers. In their study, the sensors were developed to detect adenosine and cocaine.
  • the adenosine detecting biosensor was made of nanoparticles containing three components: gold nanoparticles functionalised with 3'-thiolmodified DNA, or 5'-thiol-modified DNA, and a linker DNA molecule.
  • AuNPs gold nanoparticle-based simple readout technique was developed to detect target analytes with aptamers by naked eye (Wang et al. 2007b (supra)).
  • AuNPs have also been used in detection of small molecules, such as adenosine and potassium, by Zhang et al. (2008) (supra). Their strategy relies on the size-dependent SPR properties of AuNPs probes and aptamers.
  • a fiber-optic biosensor to detect thrombin was developed by Lee & Walt (2000) (supra).
  • the aptamers were immobilized on the surface of silica microspheres and the binding of the protein was monitored in a microarray system.
  • a similar fibre optic based aptasensor was developed by Ohk et al. (2010) (supra). Their antibody-aptamer functionalized fibre-optic biosensor can be used in the detection of Listeria monocytogenes from food.
  • the sandwich fiber-optic biosensor was based on an aptamer, specific for an invasion protein of L.
  • the antibody was immobilised on a surface to capture the target bacteria and aptamer was used as fluorescence-labelled reporter.
  • Detection methods such as for example; quartz crystal microbalance (QRM) (Liss et al., 2002 Analytical Chemistry, 74, 4488-4495; Tombelli ef a/., 2005 (supra); Hianik et al., 2007 Bioelectrochemistry, 70, 127-133), electrochemical impedance spectroscopy (EIS) (Xu et al., 2005 Analytical Chemistry, 77, 5107-51 13; Min et al., 2008 Biosensors and Bioelectronics, 23, 1819-1824; Liu et al., 2009 Electrochimica Acta, 54, 6207-621 1 ; Ho, et al., 2012
  • aptamer based biosensors Although the development of aptamer based biosensors is proceeding (Freeman et al., 2012 Analytical Chemistry, 84, 6192-9198; Kim er a/. 2012 Analytical Chemistry, 84, 6192-9198), relatively few aptasensors have been developed for the direct detection of bacterial cells, particularly pathogenic bacterial cells. Offering detection methods with little or no preanalysis preparation, coupled with the potential to detect highly pathogenic organisms, aptamers are emerging as a cost effective tool for use in rapid diagnostics for food quality and assurance.
  • a aptasensor comprising the aptamers according to the present invention.
  • the nucleic acid sequence according to the present invention is in an isolated form.
  • isolated means that the sequence is at least substantially free from at least one other component with which the sequence is naturally associated in nature and as found in nature.
  • sequence of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. Thus, for example it may be substantially free of one or more potentially contaminating polypeptides and/or nucleic acid molecules.
  • the aptamer according to the present invention is in a purified form.
  • purified means that the given component is present at a high level.
  • the component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 90%, or at least about 95% or at least about 98%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.
  • nucleotide sequence refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof).
  • the nucleotide sequence may be of synthetic or recombinant origin, which may be single-stranded whether representing the sense or anti-sense strand.
  • nucleotide sequence in relation to the present invention includes synthetic DNA and RNA.
  • the nucleotide sequence of the aptamer according to the present invention could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et a/., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T ei a/., (1980) Nuc Acids Res Symp Ser 225-232).
  • the term "fragment” as used herein may mean a portion of the aptamer sequence taught herein which has the same or better affinity, specificity or functional activity for the target of interest compared with the full sequence.
  • the fragment may be comprised of about 20 nucleotides (e.g. 18-25, preferably 19-21 nucleotides).
  • the fragment preferably has a secondary structure similar to that of the original full length aptamer over the region represented by the fragment.
  • the present invention also encompasses the use of sequences having a degree of sequence identity or sequence similarity with the nucleic acid sequence(s) of the present invention.
  • a similar sequence is taken to include a nucleotide sequence which may be at least 80%, suitably at least 90% identical, preferably at least 95 or 98% identical to the subject sequence.
  • the similar sequences will comprise the same or similar secondary structure as the subject nucleic acid aptamer.
  • a similar sequence is taken to include a nucleotide sequence which has one or several additions, deletions and/or substitutions compared with the subject sequence.
  • Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % identity between two or more sequences.
  • % identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each base in one sequence is directly compared with the corresponding base in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • % identity can be measured in terms of pure identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI package.
  • percentage identities may be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
  • CLUSTAL may be used with the gap penalty and gap extension set as defined above.
  • the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, suitably over at least 50 contiguous nucleotides.
  • the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
  • similar or homologous sequences have a similar secondary structure to the original aptamer.
  • Aptamer sequences may be refined (e.g. by random or directed mutagenesis) to alter the base sequence in order to generate aptamer molecules with greater target affinity or specificity.
  • the nucleic acid aptamers according to the present invention and for use in the present invention may include within them synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the specificity of the aptamers.
  • the present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify similar coding sequences in other organisms etc.
  • Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries. In addition, similar sequences should be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • polynucleotides may be obtained by site directed mutagenesis of characterised sequences.
  • synthetic as used herein may mean chemically synthesised.
  • the present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto.
  • hybridisation shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • PCR polymerase chain reaction
  • the present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
  • the present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
  • the present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).
  • the composition of the present invention may be used to detect a microorganism in a food.
  • the term "food” is used in a broad sense - and covers food for humans as well as food for animals (i.e. a feed).
  • the food is for human consumption.
  • the food may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.
  • the food in which the detection methods can be used may be one or more of: jams, marmalades, jellies, dairy products (such as milk or cheese), meat products, poultry products, fish products and bakery products.
  • the beverage in which the detection methods may be used include soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks, yoghurt and drinking yoghurt, calcium fortified soy/plain and chocolate milk, calcium fortified coffee beverage, wine and beer.
  • a meat based food product according to the present invention is any product based on meat.
  • the meat based food product is suitable for human and/or animal consumption as a food and/or a feed.
  • the meat based food product is a feed product for feeding animals, such as for example a pet food product.
  • the meat based food product is a food product for humans.
  • a meat based food product may comprise non-meat ingredients such as for example water, salt, flour, milk protein, vegetable protein, starch, hydrolysed protein, phosphate, acid, spices, colouring agents and/or texturising agents.
  • non-meat ingredients such as for example water, salt, flour, milk protein, vegetable protein, starch, hydrolysed protein, phosphate, acid, spices, colouring agents and/or texturising agents.
  • a meat based food product in accordance with the present invention preferably comprises between 5-90% (weight/weight) meat.
  • the meat based food product may comprise at least 30% (weight/weight) meat, such as at least 50%, at least 60% or at least 70% meat.
  • the meat based food product is a cooked meat, such as ham, loin, picnic shoulder, bacon and/or pork belly for example.
  • the meat based food product may be one or more of the following:
  • Dry or semi-dry cured meats such as fermented products, dry-cured and fermented with starter cultures, for example dry sausages, salami, pepperoni and dry ham;
  • Emulsified meat products e.g. for cold or hot consumption
  • Fresh meat muscle such as whole injected meat muscle, for example loin, shoulder ham, marinated meat;
  • Ground and/or restructured fresh meat - or reformulated meat such as upgraded cut-away meat by cold setting gel or binding, for example raw, uncooked loin chops, steaks, roasts, fresh sausages, beef burgers, meat balls, pelmeni;
  • Poultry products such as chicken or turkey breasts or reformulated poultry, e.g. chicken nuggets and/or chicken sausages;
  • Retorted products - autoclaved meat products for example picnic ham, luncheon meat, emulsified products.
  • the meat based food product is a processed meat product, such as for example a sausage, bologna, meat loaf, comminuted meat product, ground meat, bacon, polony, salami or pate.
  • a processed meat product may be for example an emulsified meat product, manufactured from a meat based emulsion, such as for example mortadella, bologna, pepperoni, liver sausage, chicken sausage, wiener, frankfurter, luncheon meat, meat pate.
  • a meat based emulsion such as for example mortadella, bologna, pepperoni, liver sausage, chicken sausage, wiener, frankfurter, luncheon meat, meat pate.
  • the meat based emulsion may be cooked, sterilised or baked, e.g. in a baking form or after being filled into a casing of for example plastic, collagen, cellulose or a natural casing.
  • a processed meat product may also be a restructured meat product, such as for example restructured ham.
  • a meat product of the invention may undergo processing steps such as for example salting, e.g. dry salting; curing, e.g. brine curing; drying; smoking; fermentation; cooking; canning; retorting; slicing and/or shredding.
  • the meat may be minced meat.
  • the food product may be an emulsified meat product.
  • meat as used herein means any kind of tissue derived from any kind of animal.
  • meat as used herein may be tissue comprising muscle fibres derived from an animal.
  • the meat may be an animal muscle, for example a whole animal muscle or pieces cut from an animal muscle.
  • the meat may comprise inner organs of an animal, such as heart, liver, kidney, spleen, thymus and brain for example.
  • meat encompasses meat which is ground, minced or cut into smaller pieces by any other appropriate method known in the art.
  • the meat may be derived from any kind of animal, such as from cow, pig, lamb, sheep, goat, chicken, turkey, ostrich, pheasant, deer, elk, reindeer, buffalo, bison, antelope, camel, kangaroo; any kind of fish e.g.
  • sprat cod
  • haddock tuna, sea eel, salmon, herring, sardine, mackerel, horse mackerel, saury, round herring, Pollack, flatfish, anchovy, pilchard, blue whiting, pacific whiting, trout, catfish, bass, capelin, marlin, red snapper, Norway pout and/or hake; any kind of shellfish, e.g. clam, mussel, scallop, cockle, periwinkle, snail, oyster, shrimp, lobster, langoustine, crab, crayfish, cuttlefish, squid, and/or octopus.
  • shellfish e.g. clam, mussel, scallop, cockle, periwinkle, snail, oyster, shrimp, lobster, langoustine, crab, crayfish, cuttlefish, squid, and/or octopus.
  • the meat is beef, pork, chicken, lamb and/or turkey.
  • the vegetable based product as taught herein may be any vegetable.
  • the vegetable based food product as taught herein may be a fermented vegetable product, a brined vegetable, or a pickled vegetable product.
  • the vegetable based food product as taught herein may be a beverage, for example a beverage containing soya such as a soya vegetable drink.
  • the vegetable based food product as taught herein may be a fermented vegetable product such as a sauerkraut fermentation, pickles from fresh, green cucumbers, fermented mixed vegetables or any fermented plant or legumes that can be for example onion, celery, beet, lettuce, spinach, broccoli, cauliflower, mushroom, potatoes, radish, cabbage, peas. It can also be silage.
  • a fermented vegetable product such as a sauerkraut fermentation, pickles from fresh, green cucumbers, fermented mixed vegetables or any fermented plant or legumes that can be for example onion, celery, beet, lettuce, spinach, broccoli, cauliflower, mushroom, potatoes, radish, cabbage, peas. It can also be silage.
  • the vegetable based food product as taught herein may be bean based, such as cheonggukjang, doenjang, miso, natto, soy sauce, stinky tofu, tempeh for example.
  • the vegetable based food product as taught herein may be grain based.
  • the vegetable based food product may be a batter made from rice and lentil (Vigna mungo) prepared and fermented for baking Idlis and Dosas, amazake, beer, bread, choujiu, gamju, injera, makgeolli, murri, ogi, sake, sikhye, sourdough, rice wine, Malt whisky, grain whisky, Vodka, batter.
  • the term "pharmaceutical” is used in a broad sense - and covers pharmaceuticals for humans as well as pharmaceuticals for animals (i.e. veterinary applications).
  • the pharmaceutical is for human use and/or for animal husbandry.
  • the pharmaceutical can be for therapeutic purposes - which may be curative or palliative or preventative in nature.
  • the pharmaceutical may even be for diagnostic purposes.
  • EXAMPLE 1 Development of novel biomolecules based on DNA (aptamers) Novel biomolecules based on DNA (aptamers) have potential applications in the area of food safety and quality assurance.
  • the method for the selection of the aptamers was developed using a previously described selective evolution technique (SELEX).
  • SELEX selective evolution technique
  • the SELEX procedure by which aptamers are generated offers the prospect of generating biomolecules with specific binding properties; similar to those exhibited by antibodies.
  • These novel molecules can be used as the basis of either simple, rapid assays or real-time monitors of food quality.
  • the application of tools based on aptamers will further help to ensure the supply of safe food and prevent incidents of food poisoning.
  • This technology also offers the prospect of the animal-free alternative to commercial diagnostic procedures that are based on the use of antibodies.
  • the selection technique was established and the detection technique developed by selecting the aptamers against non-pathogenic Escherichia coli K12. The same techinque was then used to select the aptamers against the common pathogenic food poisoning bacteria E. coli 0157, Listeria monocytogenes and Salmonella typhimurium. The aptamers for both non-pathogenic and pathogenic E. coli and for S. typhimurium were cloned and sequenced.
  • a rapid detection method based on the aptamers selected in this study can be developed or the aptamers can possibly be used as a part of an existing detection system.
  • the selection of the aptamers started with a creation of a random DNA library.
  • Nonpathogenic E. coli K12 was first used to establish the method for the aptamer selection against live bacterial cells.
  • a method based on centrifugation was used for the aptamer selection to separate the non-binding molecules from those having the affinity to the structures on bacterial cell surface.
  • the aptamers were then cloned and sequenced and the binding of these aptamer sequences were tested by using a method based on fluorescence. The binding was also visualised by a microscope.
  • a natural yoghurt containing Lactobacillus acidophilus and Bifidobacterium spp was used as an example of a food matrix to test the aptamers.
  • the yoghurt was spiked with E. coli K12 cells and the bacterial cells were roughly separated from yoghurt.
  • the aptamers were then used to detect the cells.
  • the aptamers were selected against pathogenic Listeria monocytogenes and Salmonella typhimurium.
  • the aptamers were also selected against E. coli 0157 but the selection was done from the pool of E. coli K12 binding aptamers instead of DNA library.
  • E. coli 0157 and S. typhimurium aptamers were cloned and the sequences analysed.
  • Aptamer selection and detection methods were established by selecting the aptamers against non-pathogenic E. coli K12. Aptamers were cloned and four clones were selected for the affinity tests. The sequences of these aptamers can be seen in Table 2.1. As an example, a predicted aptamer structure for aptamer 6AptK12 is presented in the table.
  • a natural yoghurt containing Lactobacillus acidophilus and Bifidobacterium spp was used as an example food matrix to test the specificity of the aptamers.
  • the yoghurt was spiked with E. coli K12 and a mixture of specific aptamers was used to detect the bacterial cells by fluorimetry analysis.
  • the fluorescence values are presented in Figure 4. It can be seen in the figure that the fluorescence is significantly higher in the E. coli K12 sample than in the negative sample where E. coli K12 has not been added to the yoghurt.
  • 3Apt223 50 5' FAM-CCCTGCAGGATCCTTTGCTGGTACCAGGGAAATCGTAGTTGATTACGATT -3' (SEQ ID No. 9)
  • typhimurium binding aptamers (2Apt223, 3Apt223 and 5Apt223) was tested.
  • the aptamers were incubated with gram negative S. enteritidis and E. coli K12 and gram positive Lactobacillus plantarum. S. typhimurium was used as a positive control.
  • the fluorescence values are shown in Figure 7. It can be seen that the aptamer 3Apt223 has the strongest binding and is the most specific aptamer from these three S. typhimurium aptamers.
  • Non- pathogenic E. coli K12 was used as a model organism for the method development.
  • a fluorimetry method was used to detect the binding of the aptamers to their target organism.
  • aptamers were selected to non-pathogenic E. coli K12 and pathogenic E. coli 0157, L. monocytogenes and S. typhimurium.
  • Aptamers against E. coli K12, E. coli 0157 and S. typhimurium were cloned and sequenced and the binding of these aptamers was demonstrated.
  • Bovine Serum Albumin (BSA) - Invitrogen, Life technologies Ltd., UK
  • FAMTM (6-Carboxyfluorescein) - Applied Biosystems, Life Technologies Ltd., UK
  • Salmonella typhimurlum - NCTC121 16 now called Salmonella enterica subsp. enterica
  • Health Protection Agency publically available from the Health Protection Agency.
  • Specific strains of Salmonella enterica serotype typhimurium are publically available and can be purchased through e.g. ATCC-LGC Standards (http://www.lgcstandards-atcc.org), such as, e.g., the strains having ATCC Number 6994, 7832, 1331 1 , 14028, 15277, 19585, 23555, 23564, 23565, 23566, 23567, 23591 , 23592, 23593, 23594, 23595, 23952, 23853, 23854 or 23855.
  • Other sources for obtaining specific strains are e.g.
  • Escherichia coli 0157 are publically available and can be obtained through for example ATCC-LGC Standards (http://www.lgcstandards-atcc.org), such as, e.g. the strains having ATCC Number 35150, 43888, 43889, 43894 and 43895.
  • Other sources for obtaining specific strains are e.g. Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(http://www.dsmz.de) or Agricultural Research Service Culture Collection (http://nrrl.ncaur.usda.gov/).
  • Oligonucleotides, sequencing and labelled aptamers were obtained from Eurofins MWG Operon. The oligonucleotides and the aptamers were analysed with Oligoanalyzer 3.1 and UNAFold (IDT - Integrated DNA technologies Inc., USA).
  • Reverse PR2 5 * -ATT CTG GGG CCC TCT AGA CTG ATT AGC GAT ACT-3'
  • PR2BIO 5' BIO-ATT CTG GGG CCC TCT AGA CTG ATT AGC GAT ACT-3'
  • PR1 FAM 5' FAM-ACC CCT GCA GGA TCC TTT GCT GGT ACC-3'
  • PR2FAM 5' FAM-ATT CTG GGG CCC TCT AGA CTG ATT AGC GAT ACT-3'
  • T7 5'-TAA TAC GAC TCA CTA TAG GG -3'
  • the stock solutions for the binding buffers were first prepared and autociaved before making up to a final volume.
  • Tris base- acetic acid- EDTA buffer (TAE)
  • PC-Agar Tryptone solution, pH 7.0
  • the tryptone solution pH was adjusted and the solution was mixed with the agar.
  • the mixture was autoclaved and plated.
  • filter sterilised antibiotic was added into the autoclaved solution (25 pg/mL, 50 pg/mL or 100 pg/mL).
  • Indicator plates Selective LB-Agar + ampiciliin, IPTG and X-Gal
  • pH of the solution was adjusted to 7.0 and 14 g of agar was added.
  • Sterilised ampiciliin 100 pg/mL, IPTG 0.5 m and X-Gal 80 pg/mL were added to autoclaved solution.
  • PCR reaction was performed by using Ready-to-Go PCR beads with 25 pmol of reverse PR1 and forward PR2 primers and 1 ⁇ of template DNA.
  • the amplification parameters were 5 min at 94°C for initial denaturation, denaturation at 94°C for 45s, annealing at 62°C for 45s, and elongation at 72°C for 45s unless otherwise stated.
  • the final elongation was 7 min at 72°C. Denaturation, annealing, and elongation were initially repeated for 15 or 20 cycles depending on the reaction.
  • the PCR products were separated on agarose gel and the products were purified from the gel with a gel extraction kit or a spin column PCR purification kit by following the manufacturer's protocols.
  • PCR samples 3 ⁇ were mixed with 6* loading dye and topped up with the water to achieve 1 ⁇ loading dye solution before the samples were applied in the wells.
  • the agarose gels were run for 40-60 minutes with an electric field of 80V to 210V depending on the size of the gel.
  • Polyacrylamide gels were run for 1 h in an electric field of 170V. The gels were visualised by UV transillumination and the pictures were taken and analysed.
  • the aptamers were selected from a pool of 100 nucleotides (nt) long DNA sequences.
  • the 100 nt sequence contained a 40 nt long random sequence and constant regions in both ends for the primer binding: 5'-ACC CCT GCA GGA TCC TTT GCT GGT ACC- 40xN TAA GAC CCC GGG AGA TCT GAC TAA TCG CTA-3'.
  • This initial ssDNA library (0.5 pmol) was amplified by PCR. The PCR products were separated on agarose gel and purified directly from the gel or with the spin columns. 2.3.4. DNA precipitation
  • Isopropanol (0.65 volumes) was added to the DNA solution (100 ⁇ ) and mixed well before the samples were centrifuged at 13,500 rpm at 4°C for 15 min. The supernatant was removed and the DNA pellet washed with 500 ⁇ of 70% Ethanol (room temperature) to remove the salt and isopropanol residues. The samples were centrifuged for 8 min at 13,500 rpm and the DNA pellet was air dried before redissolved into the TE-buffer (pH 8.0).
  • the selection of DNA aptamers against killed Francisella tuiarensis bacteria was described by Vivekananda & Kiel (2006). The selection method based on filtration was followed with some modification.
  • the double stranded DNA (dsDNA) library (2.3.3) was extracted and purified from the agarose gel and heated for 3 minutes at 94°C with an equal volume (45 ⁇ ) of binding buffer (BBf) and cooled on ice to separate the strands.
  • BBf binding buffer
  • the ssDNA samples with an equal volume of BBf (45 ⁇ ) were applied on the Multiscreen filter plates and drawn through the filter by using a vacuum manifold.
  • the samples were washed three times with 50 ⁇ of BBf and the flow through samples containing non-filter binding ssDNA sequences were collected and amplified by PCR.
  • Ready-to-go PCR beads were used with 1 ⁇ of template DNA (non-filter binding) and 25 ⁇ of each primer PR1 and PR2.
  • the amplification parameters were 5 min at 94°C for initial denaturation, denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and elongation at 72°C for 1 min. The final elongation was at 72°C for 10 minutes.
  • the samples were separated on agarose gel (2.3.2) and purified with the PCR product purification kit.
  • aptamers against live bacterial cells was first described by Hamula et al. (2008). In this study their protocol was followed with some modifications. Fresh overnight cultures of bacterial cells were used in every round of selection. Cell suspension (1 ml) was washed three times by centrifuging at 3500g for 5 min at 4°C and resuspended in 500 ⁇ of 1 x binding buffer (BB). The dsDNA was denatured to ssDNA by heating at 94°C for 5 minutes and then cooling on ice for 10 minutes. 100 ⁇ of cell suspension and 25 ⁇ of ssDNA library (PCR product) were mixed with BB containing 125 ⁇ g/ml tRNA and 0.005% BSA in order to reduce non-specific binding.
  • BB 1 x binding buffer
  • Low DNA binding (LoBind) tubes were used to reduce the aptamers to bind the tube.
  • the mixture was incubated at room temperature with gentle rotation for 45 min. Unbound aptamers were washed three times after each of the first seven incubations and five times on the eighth round of selection with 250 ⁇ of BB containing 0.05% BSA by centrifuging the cells at 4000g for 5 min at 4°C and collecting the supernatant. Bacterial cells and DNA were separated and the aptamers-containing supernatant was collected. Tubes were replaced with new fresh tubes after the first and third washes in order to eliminate aptamers which may have bound non-specifically to the tube wall.
  • the bacterial cells with bound aptamers were resuspended in 10 mM Tris-CI (pH 8.5) and the cells were heated to 94°C for 10 min to release the captured aptamers from the cells.
  • the cells were centrifuged and the aptamers containing supernatant collected.
  • the aptamer pool (supernatant) (1 ⁇ ) was amplified by PCR (2.3.1).
  • the primers and other PCR parameters were the same as those used to produce the DNA library and the PCR amplification was repeated 20 cycles.
  • Counter selection was performed in order to eliminate aptamers that bind bacteria other than the one of interest.
  • the counter selection was performed after selection round 5 and round 8.
  • the protocol for counter selection was the same as that used when selecting specific aptamers except that the unbound DNA was collected and used as a new pool of aptamers.
  • the number of PCR cycles was reduced to 15 cycles and the template concentration had to be lowered in order to obtain 100 bp PCR products.
  • the template was diluted 1 :30 and 1 :40.
  • Aptamers were labelled with biotin (BIO) or fluorescence (FAM) by amplifying the aptamer pools by PCR (2.3.1 ) using 5' biotinylated or 5' FAM-labelled primers PR1 and PR2. All PCR amplification conditions remained the same. The PCR products were separated and visualised on agarose gel and the products were purified with the spin columns as described. Before the binding reaction, aptamers were strand separated by heating the aptamers at 94°C for 10 min and cooling on ice immediately.
  • Fresh overnight bacterial culture was prepared as previously described (2.3.6) and biotin- labelled aptamers were produced (2.3.7). 100 ⁇ of single stranded aptamer solution was added to an equal volume of bacterial suspension in BB and incubated for 45 min at room temperature with gentle rotation. Unbound aptamers were washed three times by centrifuging the cells at 3500g at 4°C for 5 min and resuspending in BB containing 0.05% of BSA. New fresh tubes were changed via resuspension after the incubation.
  • the cells were resuspended in the PBS containing 0.1 % BSA and 1 g/ml peroxidase labelled Streptavidin and the samples were incubated for 45 min at room temperature in gentle rotation to allow streptavidin to bind to biotin. Unbound streptavidin was washed three times with PBS and fresh clean microcentrifuge tubes were changed after the first and third wash via resuspension.
  • the ABTS substrate (1%) in 0.05M citrate buffer in presence of 0.3% H 2 0 2 was added and after 40 min incubation the cells were centrifuged and the absorbance of the reaction mixture measured at 405 nm. Five different dilutions of aptamers (1 :2, 1 :4, 1 :10, 1 :20, 1 :40) were tested in triplicate. The reaction is illustrated in Figure 8.
  • FAM-labelled aptamer pools binding to the bacterial cell surface
  • FAM-labelled aptamers were produced (2.3.7) and strand separated by heating the aptamer pool at 94°C for 10 min and cooling immediately on ice.
  • a bacterial suspension was prepared by centrifuging of fresh overnight grown culture (1.5 ml) at 3500g for 5 min at 4°C. The bacterial pellet was then washed and resuspended in 500 ⁇ of BB. The bacterial suspension (100 ⁇ ) was incubated with denatured single-stranded aptamers for 45 min at room temperature in gentle rotation in LoBind tubes followed by centrifugation of 3500g for 5 min at 4 ° C. Unbound aptamers were washed three times with 250 ⁇ BB to remove the unbound aptamers. Incubation time was optimised to 45 min.
  • a fluorescence plate reader (495 nm, Em 520) was used to measure the fluorescence of the FAM-labelled aptamers with a sensitivity of 50. Washed bacterial cells with bound aptamers were resuspended in 100 ⁇ of BB and the samples were applied to black 96-well plates.
  • the bacterial suspension and the aptamer binding reaction were performed as described for FAM-labelled aptamers (2.3.10.1), except biotin-labelled aptamers were used.
  • Biotin-labelled aptamers were produced (2.3.7) and the aptamers were strand separated by heating at 94°C for 10 min and cooling immediately on ice.
  • the bacterial cells with biotin labelled aptamers on the surface were washed once with BB and resuspended into PBS with 1% fluorescence microspheres. The samples were incubated at room temperature for 45 min with a gentle rotation to let the fluoSpheres to bind to the biotin labelled aptamers. 1 % BSA was used to block the non-specific binding sites.
  • Bacterial cells were stained with Live/Dead SacLight kit to distinguish the aptamer binding between the live and dead bacterial cells.
  • the staining is based on CYTO-9 green- fluorescent nucleic acid stain that stains all the bacteria and the Propidium iodide red- fluorescent nucleic acid stain that only stains the bacteria with damaged membranes. FluoSpheres (2.3.10.2) were used to visualise the aptamer binding. Bacterial cells with biotin aptamers and FluoSpheres bound to them were resuspended into 0.85 % NaCI solution. An equal volume of staining solution (1 volume CYTO-9, 4 volumes Propidium iodide) was added to the suspension and incubated for 15 min at room temperature.
  • the pGEM-T Easy Vector system was used for cloning the aptamer pools. The cloning was performed by following the instructions manual (Promega Technical Manual).
  • Figure 9 is a schematic presentation of the main cloning steps. First the aptamers are PCR amplified (1. PCR) and the products purified with the spin columns. Purified aptamers are ligated to pGEM-T Easy vector by incubating in a rapid ligation buffer for an hour at room temperature (2. Ligation).
  • the linearised vector ( Figure 10) is designed with a single 3' -terminal thymidine (T) overhang at both ends.
  • the insert vectors are then transformed into the competent cells ( Figure 9, 3. Transformation) and the cells are then incubated in order to enrich the amount of the bacterial cells that have the insert vector inside (4. Cloned culture).
  • the aptamer pools 9 were PCR amplified (2.3.1) before they were ligated into the vector. Reaction components and amounts for the ligation reaction are presented in the Table below. Positive control with control insert DNA and background control without an insert were performed.
  • the ligation reactions (vectors with the inserts) were first incubated on ice with the competent cells (thawed on ice for 5 min) for 20 minutes before transformed into the JM109 competent cells.
  • the transformation of the vectors into the bacterial cells was done by heating the samples for 50 sec at 42°C and cooled down on ice for 2 minutes. Transformation efficiency was estimated by using the uncut plasmid (0.1 ng).
  • the competent cells were then incubated in SOC medium (950 ⁇ ) for 1.5 h at 37°C with 150 rpm rotation followed by the plating of the samples (100 ⁇ ) on the selective indicator plates. The plates were incubated over night at 37°C. 2.3.12.2. Analysing the positive colonies - Colour selection
  • pGEM-T Easy vector has a multiple cloning region ( Figure 11) within the a-peptide coding region of the enzyme ⁇ -galactosidase (Promega Technical manual).
  • the DNA insert deactivates the a-peptide and makes the colour screening of the recombinants possible on the indicator plates. Positive colonies were white on the plates and negative colonies were blue. Positive colonies were selected from the plates and transferred to a new selective plate. After an overnight incubation the clones were analysed by PCR and restriction analysis. 2.3.12.3. Analysing the positive colonies - PCR analysis
  • PCR Supermix HiFi was used with 25 ⁇ of both primers (PR1 and PR2) and one colony on the plate was used as a template.
  • the amplification parameters were 10 min at 94°C for initial denaturation, denaturation at 94°C for 45s, annealing at 62°C for 45s, and elongation at 72°C for 45s. The final elongation was 10 min at 72°C.
  • the PCR cycles denaturation, annealing, and elongation were repeated 20 times. PCR products were separated on agarose gel and the pictures were taken (2.3.2).
  • One positive colony from the indicator plate was incubated in 5 ml LB-broth (ampicillin 50 Mg/ml).
  • the plasmid vector was extracted from a 3 ml of overnight culture by using a plasmid extraction kit. The pure plasmid was then used for restriction analysis and sequencing.
  • EcoRI was used as a restriction enzyme to digest the insert from the plasmid.
  • the restriction map for pGEM-T Easy vector is shown in Figure 0.
  • Enzyme EcoRI (1 pi) was added to 14 ⁇ of sterile water with 2 ⁇ 10* Buffer (provided with the enzyme) and 3 ⁇ purified plasmid. The reaction was incubated at 37°C water bath for 1 h. The restriction products (9 ⁇ each) were separated on an agarose gel (2.3.2). 2.3.12.6. Sequencing of cloned vector
  • the aptamers were cloned by using pGEM-T Easy Vector system and the plasmid DNA was purified with Quick Plasmid Miniprep Kit.
  • the plasmid DNA (30 ⁇ ) samples were sent in a microcentrifuge tube to sequencing.
  • the sequencing primers (T7 and SP6r) were designed to match to vector's multiple cloning sites. The primer binding sites are marked in Figure 12 in red.
  • the first forward sequencing primer (T7) was selected from the sequencing suppliers Services a la Carte (list of standard primers).
  • the reverse primer (SP6r) was synthesised for sequencing.
  • the 100 nt long aptamer sequences were identified from the vector sequence by comparing the primers PR1 and PR2 sequences to the vector sequence. This 100 nt aptamer sequence was analysed by UNAFold program and the length of the aptamers was reduced to 35-70 nucleotides. Aptamers were synthesised with a fluorescence FAM-label and the binding was tested (2.3.9).
  • the aptamer binding to its target is dependent on its secondary structure.
  • the possible secondary structures of the aptamer sequences were analysed using OligoAnalyzer 3.1 UNAFold program. The sequence was given to the program for analysis of sodium concentration being 100 mM and magnesium concentration 1 mM as in binding buffer (BB). The temperature was set to 25 ° C. The most common secondary structure given was used as a template to reduce the length of the nucleotide sequence of the aptamers from 100 bases to 35 to 70 bases. The analysis of these aptamers was done with the UNAFold.
  • Aptamers are single-stranded DNA or RNA ligands that can be selected to bind to proteins but also smaller molecules such as organic dyes (Ellington & Szostak, 1990) as well as prions (Iqbal et al., 2000), bacterial cells (Hamula et al., 2008) and viruses (Symensma et al., 1996). Binding of the aptamers to their target mainly encompasses all types of non-covalent binding except normal standard nucleic acid bond formation (Watson-Crick base pairing).
  • the counter bacteria used were Lactobacillus bulgancus after selection round 5 and Bacillus subtilis and Salmonella typhimurium after selection round 8.
  • the aptamer pool 9 was biotin labelled and the binding of the aptamers was tested with an enzyme linked technique. 3.2. METHODS
  • a random DNA library was produced (2.3.3).
  • the PCR cycle (denaturation-annealing- elongation) was initially repeated over 30 cycles with 1 min reaction times resulting in the formation of non-specific products.
  • Primers were analysed with an Oligoanalyzer to see the primer-dimers that can possibly be formed.
  • the reaction times, temperatures, template concentration and the number of PCR cycles were optimised.
  • the PCR products were separated on agarose gel (2.3.2) and the pictures were taken.
  • the DNA library was purified from gels with a gel extraction kit. 3.2.2. Selection of E. coli K12 specific aptamers
  • the aptamers were selected from a random DNA library to bind specifically to nonpathogenic strain E. coli K12. Filtration selection (2.3.5) was first used to select the aptamers but was found to be overly complicated. The elution of the bound aptamers was difficult, as boiling elution buffer with high urea concentration was needed. It was impossible to add the buffer that was boiling on the filter. The other major problem occurred when the elution buffer was boiled for too long and some of the water was evaporated. This resulted in a precipitation of the urea on a filter making the aptamers impossible to elute.
  • the EDTA in the elution buffer keeps the DNA more stable when stored but the DNA had to be precipitated, purified and redissolved in another buffer for next steps of the selection.
  • the first 5 aptamer pools were selected (2.3.6) and the pools were amplified by PCR (2.3.1) using 20 reaction cycles.
  • the first counter selection was performed after the fifth round of selection where L bulgaricus was used as a counter bacterium.
  • the samples were collected and amplified by PCR. After the counter selection the PCR was optimised.
  • the template was diluted to 1 :30 and the number of amplification cycles was reduced to 15 cycles.
  • the template control was performed to see if the addition of a template to the reaction appears as a band on an agarose gel.
  • Aptamer pool 5, that has gone through the counter selection was used as an aptamer pool for selection round 6. Samples were amplified by PCR with 15 PCR cycles.
  • Selection round 7 was performed as normal and both replicates were amplified by PCR twice in two separate tubes due the low PCR product yield after the previous selection round. Those two PCR products were extracted and mixed together and aptamer pool 8 was selected from this pool of aptamers in two replicates. The number of PCR cycles was increased to 20 cycles as 15 cycles did not result in a PCR product that could have been seen on an agarose gel. This increase of the PCR cycles resulted in a higher number of aptamer copies and therefor the PCR product can be visualised on an agarose gel. Two replicates were amplified in two separate reaction tubes and mixed together.
  • the second counter selection was done by selecting the aptamers that are not binding to B. subtilis or S. typhimurium.
  • Aptamer pool 8 was used for the selection and the non-binding samples were collected and amplified by PCR.
  • the template for the PCR was diluted 1 :40 and 15 rounds of PCR was used.
  • the ninth pool of aptamers was selected from the pool 8. Pool 9 was PCR amplifies and separated on polyacrylamide gel (2.3.2). Polyacrylamide gel was used for these samples in order to see which one, agarose or polyacryamide gel, is more suitable for separating the aptame PCR products.
  • the biotin-labelled aptamer pool 9 selected to bind E. coli K12 was incubated with bacterial culture (2.3.8). Bacterial cells with biotin-labelled aptamers bound to them were incubated with streptavidin peroxidase following the addition of ABTS and H 2 0 2 . The colour change was detected by measuring the absorbance of the samples at 405 nm. Five different dilutions of aptamers (1 :2, 1 :4, 1 :10, 1 :20, 1 :40) were tested in triplicate. The results were analysed with the analysis of variance (ANOVA). 3.3. RESULTS AND DISCUSSION
  • the random DNA library was produced by PCR and the samples were separated on agarose gel. When the PCR amplification reaction was repeated 30 cycles, and the reaction (denaturation, annealing and elongation) times were 1 min, non-specific products were observed. In these conditions non-specific PCR products were appearing on agarose gel pictures as extra bands ( Figure 13). In lane 0 is a PCR control, where only PCR primers PR1 and PR2 are added, products smaller than 100 bp and a faint 100 bp band can be seen. In lane 1 where the template DNA is added, two bands can unexpectedly be seen. These extra bands can be caused by the dimerisation of the primers, for example, formation of self- dimers or hetero-dimers.
  • PCR products without non-specific products were achieved when the number of the PCR cycles was reduced to 15 or 20 and the reaction times were reduced from 1 min to 45 s.
  • PCR control sample appears to be clear and non-specific bands cannot be observed on gel pictures.
  • the DNA library was produced by using these conditions and the agarose gel of the library is shown in Figure 15.
  • the samples (DNA library) are on gel in lanes 1-6 and can be seen as thick bands.
  • the PCR products were expected to be 100 bp in size because the DNA library size was 100 nucleotides.
  • the bands on the gel seem to be 140 bp instead of 100 bp. It has been found that the pre-stained agarose gels might affect the mobility of DNA on gel and especially small DNA fragments might be affected (Miller et al.
  • E. coli K12 specific aptamers were selected to bind non-pathogenic strain E. coli K 2 by using a centrifugation method first described by Hamula et al. (2008) with some modifications. Nine rounds of selection and counter selection after selection round 5 and 8, were performed. Agarose gels after the selection round 1 , 2, 3 and 4 are presented in Figure 16. The aptamer pools can be seen in the gel images in lanes 1 and 2 as 100 bp or slightly bigger bands. No amplification can be seen in DNA control samples where no bacterial cells were added (lanes 3), as expected. This is because the aptamers have been washed off from the samples as there are no binding sites for them in the solution.
  • the aptamers are not binding anything else such as the tube wall.
  • the bacterial control samples in lanes 4 are clear. This shows E. coli K12 has no DNA sequence for the PCR primers used in this experiment to bind and therefore cannot be amplified in PCR.
  • the PCR control samples in lanes 0 have no 100 bp bands, only a faint band (smaller than 50 bp) that is a primer dimer, as expected.
  • the counter selection was performed by using L. bulgaricus as a counter bacterium.
  • the agarose gel pictures are presented in Figure 17.
  • the PCR product of aptamer pool 5 is on gel a ( Figure 17a) in lanes 1 and 2 and the aptamer pool 5 after the counter selection is on gel b ( Figure 17b) in lanes 1 and 2.
  • the counter selection products are the aptamers that are binding E. coli K12 but not L. bulgaricus.
  • a template control in lane 1 on gel b ( Figure 17b) was performed to see if the addition of a template can be seen on an agarose gel. As expected, no 100 bp template band can be seen and this indicates that the bands seen on gel images are PCR amplification products.
  • Lanes 0 on the gels are PCR control samples where the primers were added with no template DNA. All these control samples are clear as expected. The faint bands around 50 bp are the primers.
  • Agarose gel pictures of aptamer pools 6 and 7 are presented in Figure 18. The aptamer pool 6 has faint bands on the gel a ( Figure 18a). Because of the small yield of the PCR product 6, two aptamer pool 7 samples were amplified in two replicates. Samples 1.1 and 1.2, and samples 2.1 and 2.2 on gel b ( Figure 18b) were mixed together after they were purified in order to achieve an aptamer pool with more aptamers. On lanes 0, where the PCR control samples are, no amplification can be seen because no template DNA was added.
  • the aptamer pool 9 was selected and the PCR products were separated on polyacrylamide gel. Polyacrylamide gel was used to see if it is suitable for separating aptamer samples.
  • the gel picture of the aptamer pool 9 and the control samples are presented in Figure 20. Two bands can be seen on the gel in lanes 1 and 2 around 100 bp. These samples are aptamer pool 9 in two replicates. It can be seen that the molecular weight marker (lane M) has unusual bands when comparing the bands to the other gel images above. It seems that the ladder used in this experiment is not suitable for polyacrylamide gels.
  • the bacterial control sample in lane 3 and the DNA control sample in lane 4 are clear, as expected.
  • the bacterial control sample only contains bacterial cells and the DNA control sample only contains the DNA aptamers that have been washed off because there were no binding sites for the aptamers in the mixture.
  • PCR control sample where only the primers have been added, is on lane 0. No amplification can be detected, as expected. The faint bands at the bottom of the image are the primers.
  • the polyacrylamide gel can be used for separating the samples, but agarose gels were used in further experiments because of their easiness.
  • the biotin-labelled aptamer pool 9 was produced by PCR. The labelling was done by using biotinylated primers. The PCR products were separated on agarose gel to see the size of the products.
  • biotin-labelled aptamer pool 9 selected to bind E. coli K12 is presented. Aptamer pools are the 100 bp bands in lanes 1 -12. In lane 0 the PCR control sample where no template DNA was added is clear, as expected. Samples were purified with spin columns and used for the binding reaction.
  • EXAMPLE 4 Development of fluorescence based detection method for Escherichia coli K12 binding aptamers
  • the method for selecting aptamers against live bacterial cells was developed.
  • the specific aptamers were selected to bind to non-pathogenic E. coli K12 by using a method based on centrifugation (3.2.2).
  • the binding of these aptamers to their target was demonstrated with an enzyme linked method (3.3.4), where the aptamers were first labelled with biotin.
  • biotin As the strong binding between streptavidin and biotin is well known, peroxidase-labelled streptavidin was attached to biotin. Addition of a substrate led to a colour change when reacting with the peroxidase. This colour change correlates to the amount of aptamers bound to the bacterial cell surface.
  • This method is based on fluorescent-labelled streptavidin beads that can bind to biotin labelled-aptamers and can possibly be used to test the aptamer binding to smaller and faster moving bacterial cells such as Salmonella. This study demonstrates the E. coli K12 aptamers binds specifically to its target.
  • Aptamers were selected to bind to E. coli K12 (3.2.2).
  • the aptamer pools used in the experiments were labelled with 5' fluorescence (FAM) label (2.3.7) and by detecting the label the aptamers will be detected.
  • aptamer pools were produced by PCR using the aptamer pool 9 as a template. More template was produced by amplifying the aptamer pool 9 PCR product in dilution 1 :40 (Nested template) by PCR. The PCR amplified aptamer pools were separated on agarose gels and the images were taken followed by a purification of the product by spin columns. The PCR product concentration was measured using a spectrophotometer to measure the absorbance of the samples at a wavelength 460 nm. 4.2.1.2 Fluorimetry
  • Fluorescence values of the E. coli K12 bacterial cells when FAM-labelled aptamers were bound to them were measured to detect the aptamer binding.
  • E. coli K12 binding aptamers with FAM-labels were incubated with bacterial cells (2.3.9.1 ) at three different concentrations (10 pmol, 20 pmol and 30 pmol). Samples were incubated at room temperature for one hour and the samples were washed with binding buffer (BB). The fluorescence of the samples was measured with a fluorescence plate reader (2.3.9.3) and the results were analysed with ANOVA. 4.2.1.3 Fluorescence microscope
  • the fluorescence microscope was used to visualise the fluorescent labelled aptamers bound to bacterial cell surface.
  • E. coli K12 binding aptamers with FAM-labels were incubated with overnight grown E. coli K12 culture (2.3.9.1 ) and the samples were visualised under a fluorescence microscope (2.3.9.2).
  • Two different concentrations (10 pmol and 50 pmol) were used and a control with no aptamers.
  • the images were taken from six random fields with green (495 nm) and visible light and the fluorescent labelled bacterial cells were counted from the images. Results were analysed with a statistic test the analysis of variance (ANOVA). 4.2.2 Optimal binding time of the aptamers
  • FAM-labelled aptamer pool 9 specific for E. coli K12 was used to measure the optimal binding time of the aptamers.
  • the binding reaction (2.3.9.1 ) was performed with an aptamer pool 9 (10 ⁇ , approximately 6 pmol) and 90 ⁇ of an overnight culture.
  • the samples were incubated at room temperature in triplicate for 0, 15, 30, 45, 60, and 75 minutes and the fluorescence values were measured with a fluorescence plate reader (2.3.9.3).
  • the samples were washed three times with 200 ⁇ of BB and the fluorescence was measured from the washes in order to see how many washes were needed to wash off the nonbinding aptamers.
  • the fluorescence results were analysed with the ANOVA. 4.2.3. Binding of the aptamer pool 3, 5, 7 and 9
  • Binding of the aptamer pools was tested in order to see their binding capacity. Aptamer pools were collected after each round of aptamer selection (3.2.2) and the FAM-labelled aptamer pools were produced by PCR. Binding properties of the aptamer pools 3, 5, 7 and 9 were tested by incubating the aptamers with bacterial cells (2.3.9.1) and measuring the fluorescence of the samples. Aptamers were incubated in 50 ⁇ of E. coli K12 suspension with three aptamer concentration (10 pmol, 15 pmol and 25 pmol) for aptamer pool 5 and 9.
  • aptamers Due the small number of aptamers (PCR product), only two different aptamer concentrations (10 pmol and 15 pmol) were used for the analysis of aptamer pool 3 and 7. After 45 minutes incubation at room temperature the bacterial cells were washed and the fluorescence of the samples was measured with a fluorescence plate reader (2.3.9.3).
  • the specificity of the aptamers was tested in order to see if the E. coli K12 binding aptamers are specific to E. coli K12 or if they bind to other bacteria too.
  • FAM-labelled aptamer pool was incubated with different bacterial cells. Two different types of experiments were performed. The samples were visualised under a fluorescence microscope and the fluorimetry experiments were performed. The aptamers were also tested to see if E coli K12 could be detected from a mixture of bacterial cells.
  • E. coli K12 specific aptamer pool 9 was tested with E. coli B, B. subtilis and S. aureus.
  • E. coli K 2 was used as a positive control.
  • Bacterial suspensions were prepared (2.3.9.1 ) and 50 ⁇ of this suspension was incubated with three different amounts of aptamers.
  • E. coli K12 was incubated with 10 pmol, 20 pmol and 30 pmol of aptamers and the other strains (E. coli B, B. subtilis and S. aureus) with 5 pmol, 20 pmol and 30 pmol of aptamers.
  • 5 pmol of aptamers were used instead of 10 pmol, because not enough aptamers were produced (small PCR yield).
  • the aptamer pool nine was tested to see if the aptamers are able to detect the E. coli K12 bacterial cells from a mixture of different bacterial cells.
  • the bacterial mixture containing equal amounts of E. coli K12, E. coli B and S. aureus was prepared.
  • Each bacterial suspension was prepared as described (2.3.9.1 ) and each suspension was mixed together to a total sample volume of 100 ⁇ .
  • the control samples were prepared by adding a third of bacterial suspension and topped up to 100 ⁇ with BB.
  • the aptamers (20 pmol) were incubated with the bacterial cell suspension followed by the washes. The fluorescence was measured by a plate reader (2.3.9.3).
  • 4.2.4.3 Binding of the aptamers to L. acidophilus
  • L acidophilus is a common bacterium found in dairy products such as yoghurt and was therefore chosen to be one of the strains to be tested.
  • the specificity experiment (4.2.4.1) was performed by incubating 20 pmol of FAM-labelled aptamers with a L. acidophilus strain. E. coli K12 was used as a positive control. The negative control samples, with no added aptamers, were made for both strains. The fluorescence values were measured by a fluorescence plate reader (2.3.9.3). 4.2.5 Fluorescence microspheres
  • the Live/Dead SacLight staining was introduced to see if the E. coli K12 specific aptamers are binding to live or dead bacterial cells (2.3.11).
  • the FAM-labels of the aptamers were difficult to see on the microscope images because of the brighter fluorescence of the Live/Dead BacLight staining.
  • the FluoSpheres were used to detect the bound aptamers.
  • the biotin labelled aptamer pool was first incubated with E. coli K12 as previously described (4.2.5). Once the aptamers and the fluorescence microspheres were bound to the bacterial cell surface the Live/Dead staining was performed (2.3.1 1). The samples were visualised by a fluorescence microscope and the images were taken.
  • E. coli K12 binding FAM-labelled aptamer pool 9 was performed by fluorimetry using a fluorescence plate reader.
  • the fluorescence of the E. coli K12 samples incubated with different aptamer concentrations (10 pmol, 20 pmol and 30 pmol) are shown Figure 23.
  • These results show that the aptamers have bound to live E. coli K12 cells.
  • This method developed for aptamer detection is easy to perform, repeatable and will be used in further aptamer characterisation experiments.
  • Fluorescence microscopy was used to visualise the bacterial cells with aptamers bound to them. The images were taken from each field with fluorescence and visible light. Images of negative control samples with no added aptamers (0 pmol) and samples with 10 pmol and 50 pmol aptamers are shown in Figure 24. The results show that when FAM-labelled aptamers are not added (0 pmol) (left hand side), no fluorescent dots can be seen but the dots can be seen when aptamers are added (10 pmol and 50 pmol), as expected. This indicates that the aptamers have bound to the live E. coli K12 cells.
  • Optimal binding time of the aptamers was measured by incubating the aptamer pool 9 with E. coli K12 and measuring the fluorescence after different time points. The first sample collected was after 0 minutes of incubation and then samples were collected every 15 minutes until 75 minutes. The samples were washed three times before the fluorescence was measured. The fluorescence values are presented in Figure 27. It can be seen that the highest fluorescence values (best binding of the aptamers) is achieved after 45 minutes incubation. Next sample collected (60 min) is having a lower fluorescence. For the 75 min sample the fluorescence increased again back to the same level with the 45 min sample.
  • the fluorescence values for non-binding aptamers after the first wash are presented in Figure 28, and after the second and third wash in Figure 29. It is noticeable that the fluorescence values are very high (fluorescence 290-330) after the first wash ( Figure 28) comparing to the second or third washes (fluorescence less than 4) ( Figure 29). This indicates most of the non-binding aptamers are washed off after the first wash and three washes is enough to wash off the non-binding aptamers.
  • Another reason for the low binding of the aptamers can be that there are not enough bacterial cells serving the binding sites for the aptamers. It has previously been demonstrated that these E. coli K12 binding aptamers do not detect all of the E. coli K12 cells in the solution ( Figure 24). In Figure 28 it can also be seen that less fluorescence was detected in the 45 min sample than in the other samples. This may indicate that more aptamers have bound to the bacterial cell surface and have not been washed off. In conclusion, 45 minutes was demonstrated to be an optimal binding time for the aptamers and three washes can be used to remove non-binding aptamers from the solution.
  • Aptamers were selected to bind to live E. coli K12 bacterial cell by repeating the selection process nine times. The binding of the selected aptamer pool 9 was previously demonstrated. In this study the binding of the previous aptamer pools were tested in order to see if the binding increases during the selection process. FAM-labelled aptamer pools 3, 5, 7 and 9 were incubated with E. coli K12 bacterial cells and the fluorescence of the samples was measured. The fluorescence values of each pool are presented in Figure 30. Aptamer pool 5 and 9 were tested with three concentrations (10 pmol, 15 pmol and 25 pmol) while aptamer pools 3 and 7 were tested with two different concentrations (10 pmol and 15 pmol).
  • PCR yield (aptamers) was smaller for the aptamer pool 3 and 7.
  • the PCR yield of different aptamer pools might vary depending on the quality of the template or the amount of the aptamer molecules in each sample.
  • the results show that the highest values were obtained for pools 7 and 9. This indicates that at least seven or nine rounds of selection have to be performed in order to achieve a specific pool of aptamers. If aptamers were further selected, for example aptamer pool 10, it could be possible to maintain even more specific pool. In this study, pool 9 was selected to be enough specific to bind to £. coli K12 bacterial cells. 4.3.4 Specificity of the £. coli K12 binding aptamers
  • the fluorescent labelled aptamer pool 9 was incubated with £. coli K12, £. coli B, B. subtilis and S. aureus cultures with three different concentrations. For £ coli K12 10 pmol, 20 pmol and 30 pmol aptamers were used and for £. coli B, B. subtilis and S. aureus) 5 pmol, 20 pmol and 30 pmol aptamers were used. The fluorimetry test was performed for all samples. The fluorescence values are presented in Figure 31. In the figure it can be seen that the more aptamers there are in the sample the higher the fluorescence.
  • the highest fluorescence values can be seen in £ coli K12 sample, even when small amounts (5-10 pmol) of aptamer are added.
  • the fluorescence values for the other samples (£. coli B, S. aureus and B. subtilis) are very low.
  • B. subtilis has given a higher fluorescence value when 30 pmol aptamers have been added.
  • the higher fluorescence might not be actual binding to bacterial cells and can possibly be caused by the clusters the bacteria forms while growing in broth.
  • the aptamer concentration 20 pmol was selected to use in the following specificity experiments because it shows high fluorescence for £. coli K12 but not much fluorescence for £ coli B, S. aureus or B. subtilis.
  • the fluorescence microscope images were taken of each sample (£. coli K12, £ coli B, S. aureus and B. subtilis) from five random fields and the fluorescent dots were counted. The images were taken with a green fluorescence light and with a visible light.
  • the microscopy images of 20 pmol samples of £. coli K12 (positive control) and E. coli B are presented in Figure 32 and of B. subtilis and S. aureus are presented in Figure 33. It can be seen in the microscope images, when the FAM-labelled aptamers are added, that bacterial cells with fluorescent labels are visualised as green dots. When the FAM aptamers were incubated with £. coli B, four fluorescent dots can be detected in the image.
  • the capability of the aptamer pool to detect E. coli K12 from a mixture of the bacterial cells was tested.
  • FAM-labelled aptamers were incubated with a mixture of bacterial suspension (E. coli K12, E. coli B and S. aureus) and each suspension individually.
  • Figure 35 the fluorescence values of the samples are presented. In the diagram, it can be seen the highest fluorescence was detected from the mixture of these three bacterial suspensions (Mix) as expected. Minor fluorescence can be detected from E. coli B and S. aureus samples.
  • the positive control sample (E. coli K12) has the strongest fluorescence, as expected. There is no big difference between the fluorescence values of the mixture sample and the positive control sample.
  • Fluorescent labelled aptamers binding to smaller bacterial cells than E. coli K12 might be difficult to detect under the microscope. Fluorescence microspheres technique was developed to detect the aptamer binding to small bacterial cells. Biotin labelled aptamer pool 9 was produced and the aptamers were incubated with E. coli K12 bacterial cells. The FluoSpheres were added and samples were incubated in order to let the microspheres bind to biotin label of the aptamers. The samples were visualised under the fluorescence microscope and the images were taken. Examples of the microscope images are shown in Figure 37. The bacterial cells can be seen in the images as well as the microspheres because the images were taken with a green fluorescence light and a visible light.
  • the Live/Dead SacLight staining was performed in order to see if live or dead E. coli K12 bacterial cells are detected with the aptamers.
  • the FAM-labelled aptamers were first incubated with E. coli K12 and the Live/Dead staining was performed. The FAM-labels were difficult to see because of the brighter colour from Live/Dead staining.
  • Figure 38 a fluorescence microscope image is shown. It can be seen that it is impossible to detect if the aptamers have bound to green, live bacterial cells. On red dead cells some green colour can be detected but this colour might also be derived from the Live/Dead staining kit where the colour has partly stained the bacterial cells.
  • the biotin-labelled aptamer pool 9 was then incubated with E. coli K12 bacterial cells and the FluoSpheres were attached to them followed by Live/Dead staining of the cells.
  • the microscope images are presented in Figure 39. It can be seen that some of the fluoSpheres have bound to bacterial cell surface on negative control samples (-) where biotin labelled aptamers have not been added. This indicates that the fluoSpheres might bind to bacterial cell surface. Not much binding can be detected on the sample (+) where the biotin labelled aptamers have bound to bacterial cells surface. Some of the molecules could have been washed off during the staining. Also, the bond strength between the aptamer and the bacterial cell in not known and it is possible that this bond breaks when the non-binding FluoSpheres are washed off. The Live/Dead staining can also affect to the binding.
  • Aptamers were selected to bind specifically to E. coli K12 live bacterial cells. The binding was first detected with a method based on an enzymatic reaction. This method was time consuming and the results were unreliable. The easier and more reliable fluorescence based method was developed and by using this method the binding properties of the aptamers were analysed.
  • the binding of the fluorescent-labelled aptamers was tested by comparing the fluorescence values measured. This method developed makes the testing of the aptamer binding easier and allows to comparing different samples to each other.
  • the fluorescent labelled aptamer binding can also be visualised under a fluorescence microscope. However, this method cannot be used for statistical analysis, is time consuming and is not very reliable as the bacterial cells are not spread consistently over a microscope slide, but it gives interesting visual information about the aptamer binding.
  • the optimal binding time for the aptamers is 45 minutes and the binding is not significantly improving when the incubation time was increased. Three washes are enough to wash off the non-binding aptamers.
  • An alternative method was introduced where the same principles of visualising the samples under the fluorescence microscope were used. In this experiment the aptamers were labelled with a biotin-label and fluorescent labelled microspheres were attached to them. The samples were then visualised under the microscope. The method tested in this study shows the aptamer binding can be detected with the fluorescence beads.
  • the aptamers have been selected to bind live E. coli K12 bacterial cells. The binding has been demonstrated by an enzymatic method (3.3.4) and by fluorescence based methods (4.3.1). The selected aptamers are specifically binding to E. coli K12.
  • the aptamer pool 9 which is specific to £ coli K12, was cloned with a commercial cloning kit and competent bacterial cells. The clones were analysed and the cloned plasmids were extracted and sequenced. From the plasmid vector sequence the aptamer sequence was identified and analysed. Since the synthesis of long (100b) nucleotide sequences is being difficult and expensive, the nucleotide sequences of the aptamers are often reduced. James (2007) stated that the reduction below 60 nt is almost essential for efficient chemical synthesis and a size of 40 nt or below is desirable on grounds of cost of goods.
  • the shortened aptamer sequences were isolated and synthesised with a fluorescent label.
  • the fluorescence based detection methods, fluorimetry and fluorescence microscopy, were used to detect the aptamer binding to the bacterial cells.
  • the specificity of the aptamers was tested by incubating the fluorescent aptamers with E. coli B and S. aureus followed by fluorescence measurements. Once the aptamer sequences are identified the aptamers can be chemically synthesised.
  • aptamer pool 9 The binding of aptamer pool 9 to E. coli K12 has been demonstrated (3.3.4; 4.3.1 ). So far, the aptamer pools with unidentified sequences have been produced by PCR. In order to synthesise the aptamers the individual sequences need to be determined. This was done through cloning of the aptamers.
  • the £. coli K12 aptamer pool 9 was produced by PCR (3.3.2) and ligated into a pGEM-T easy vector using a method described in 2.3.12.1. It has been suggested to use 1 :1 insertvector ratio for the control insert DNA for pGEM-T easy vector while a optimisation of the ligation reaction for aptamers was recommended (Promega Technical Manual).
  • the inser vector ratio was optimised for ratios 1 :4, 1 :2 and 1 :1.
  • the PCR product (insert) concentration was estimated to 0.8 ng/ ⁇ and the PCR product size was 100 b (0.1 kb).
  • the pGEM-T easy vector (50 ng) size is 3 kb. Therefore, the PCR product amounts for this reaction were 0.4 ng (1 :4), 0.8 ng (1 :2) and 1.7 ng (1 : 1).
  • the ligation reaction components are listed in the Table below.
  • the transformation of the ligation reaction (2 ⁇ ) into the competent cells was done as described in section 2.3.12.1. After the transformation the samples were incubated in SOC medium before plating 100 ⁇ of suspension on LB plates with ampicillin/IPTG/X-gal. The colonies were counted from overnight grown selective plates (2.3.12.2). From the selective plates, eight positive white colonies were randomly selected and streaked on a new indicator plate. The plates were incubated overnight.
  • the colonies on the selective plates were analysed to see if the ligation and transformation had worked.
  • the PCR analysis was performed for eight samples (CI1 -CI8) from the overnight selective plates with PCR Supermix HiFi (2.3.12.3) and the aptamer primers PR1 and PR2.
  • the templates used in this reaction were taken directly from the colonies.
  • the initial denaturation of PCR cycle breaks down the bacterial cell walls releasing the plasmid DNA with an insert.
  • the primers used are the same as the primers used for aptamer production. If the cloning of the insert (aptamer) and the transformation of the vector inside the bacterial cells have been successful, a 100 bp long band should be visible on the agarose gel images.
  • the PCR control no template DNA
  • a plasmid vector control template 0.5 ⁇ vector
  • the plasmid DNA control shows if the plasmid itself has binding sites for the primers used in this reaction.
  • the PCR products were separated on an agarose gel.
  • the cloned eight colonies were also tested with a restriction analysis to see if the cloning has worked. Restriction analysis (2.3.12.5) was performed to confirm the results of PCR analysis. The colonies were inoculated into LB-media and the plasmid was purified from the overnight grown culture (2.3.12.4). EcoRI enzyme has digestion sites in the both sides of the insert and therefore was selected to digest the insert out from the plasmids (CI1-CI8). The digestion sites can be seen in the sequence of the pGEM-T Easy vector in Figure 1 1. The samples were separated on an agarose gel (2.3.2). 2 Binding of the cloned aptamers to E. coli K12
  • CM and CI2 Two cloned colonies (CM and CI2) were randomly selected to test if they are binding to E coli K12.
  • the clones were produced by PCR (2.3.1 ) under the same conditions using the fluorescent FAM-primers as in the usual aptamer production.
  • the template used in this reaction was a nested template for the cloned aptamers in dilution 1 :60.
  • the PCR products were separated on agarose gel followed by purification of the PCR product.
  • the overnight grown E. coli K12 culture was prepared as previously described (2.3.9.1 ).
  • the bacterial suspension (100 ⁇ ) was incubated with 10 pmol, 20 pmol and 40 pmol of aptamers.
  • the samples were washed and the fluorescence was measured by the plate reader (2.3.9.3).
  • the cloned aptamers were sequenced directly from the plasmid vectors. According to the results of the PCR analysis and the restriction analysis four positive samples were selected for the sequencing. Plasmid DNA samples CI1-CI4 (30 ⁇ each) were sequenced (2.3.12.6). The aptamer sequences were identified from the vector sequence by identifying the primers binding sites of the primers PR1 and PR2 from the vector sequence. Between the primer sequences the aptamer sequence with 40 nucleotides long random sequence were found. The aptamer binding to its target is dependent on its secondary structure. The possible secondary structures of the aptamer sequences were analysed as previously described (2.3.12.7).
  • the binding properties of the sequenced aptamers were tested. Aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 were synthesised with 5' FAM-label and the binding to E. coli K 2 was tested. The overnight grown E. coli K12 culture (2.3.9.1) was washed and incubated with the aptamers (10 pmol, 20 pmol, 50 pmol and 100 pmol) in triplicate. Due the synthesis of single stranded molecules the denaturation of the aptamers is not necessary. The aptamers (10 ⁇ ) were added to 100 ⁇ bacterial suspension in BB. The fluorescence of the samples was read by the plate reader (2.3.9.3). The values were tested with the analysis of variance (ANOVA). To visualise the binding of the aptamers, 50 pmol samples were selected and visualised under the fluorescence microscope (2.3.9.2).
  • the binding of the aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 to E. coli B and S. aureus was tested.
  • E. coli K12 was used as a positive control.
  • a mixture of the aptamers (50 pmol) was used in the binding reaction (2.3.9.1 ) in triplicate. The samples were incubated and after the washes the fluorescence was measured by the plate reader (2.3.9.3) and the samples were visualised under the fluorescence microscope (2.3.9.2). The fluorescence values were analysed using the statistic test ANOVA.
  • the same protocol was then repeated with individual aptamers (20 pmol) in order to see the differences between different aptamers. Due the problems with the plate reader the sensitivity had to be changed from 50 to 75 in order to read the plate.
  • aptamer pool 9 was amplified by PCR and cloned with pGEM-T Easy vector and JM109 competent cells. The colonies on indicator plates were counted and a number of positive (white) and negative (blue) colonies is presented in the Table below. The number of colonies on the plates was small.
  • the positive control sample was spread on a selective plate, where the ampicillin used was not fresh and therefore might have lost its activity. On a normal selective plate the positive control sample express the colonies if the transformation has been successful. Due the inactive ampicillin the plates were full of colonies.
  • the positive colonies are only growing on the plates due the ampicillin resistance of the bacteria. Even though the positive control sample was full of colonies, further analyses of the actual samples were performed. Eight white colonies were selected from the plate and streaked on new selective plates. After an overnight incubation on selective plates, five colonies (CI3, CI4, CI6, CI7 and CI8) out of eight expressed the blue colour while three remained white (CM , CI2 and CI5). The blue colour may be expressed even if the insert has not been ligated into the vector. In a normal situation where the insert has successfully been ligated the reading frame for the lacZ gene has been interrupted.
  • the colonies from the selective plates were analysed to see if the insertion of the aptamers into the vector has been successful.
  • the PCR analysis was performed for the cloned colonies.
  • the aptamer primers PR1 and PR2 were used in this experiment. Therefore, only the colonies with the aptamer sequence and the primer binding sites are amplified in PCR resulting in a 100 bp products. If 100 bp long bands can be seen on a gel the aptamer cloning has been successful.
  • the agarose gels of the PCR amplification products are presented in Figure 40. It can be seen that the samples CM , CI2, CI3 and CI4 have a 100 bp band and a very faint band can be seen in sample CI6.
  • the pGEM-T Easy vector bands (Plasmid DNA) and the digested inserts (aptamers) are in lanes CM - CI8. It can be seen that the plasmid purification has been successful in all of the samples. The large plasmid DNA has not moved far on the gel and the range of the molecular weight marker does not reach to it. Also the intensity of the plasmid DNA bands is high when compared to the intensity of the insert bands. The lighting of the image has been changed in order to get the faint insert band visible. The insert band can be seen in all of the samples except CI1 and the band for CI2 is very faint and can hardly be detected.
  • the aptamers were produced using the usual aptamer production method. Two different cloned aptamers were used as a template. The agarose gel of the PCR products is presented in Figure 42. CI1 and CI2 cloned aptamers were produced with FAM-labels. The PCR products are on lanes CM and CI2. All samples have a strong 100bp band. In lane 0 is the PCR control sample where only primers were amplified without a template. No amplification products can be seen in this lane as expected. The faint band ( ⁇ 50 bp) in this lane is the primer dimer. The PCR products were purified with spin columns.
  • the purified FAM-labelled aptamers were incubated with E coli K12 using the aptamers in three different concentrations (10 pmol, 20 pmol and 40 pmol).
  • the fluorescence of the samples was measured using a plate reader and the fluorescence values are shown in Figure 43. It can be seen in Figure 43 that the fluorescence is higher when more aptamers have been added. This shows that the cloned aptamers can bind to E. coli K12 bacterial cells.
  • the higher fluorescence levels can be detected from sample CM than the sample CI2. It is possible that this aptamer binds more strongly to the target than sample CI2. It is possible that these two different samples would have produced curves closer to each other if the replicates had been performed. .3 Sequencing of cloned vectors and aptamer analysis
  • the aptamer pool 9 specific to £ coli K12 has been demonstrated to be binding to its target.
  • the aptamers in this pool are still unknown and to obtain the aptamer sequences the cloned aptamers need to be sequenced.
  • Four positive colonies were selected and the plasmid vectors were extracted.
  • the aptamer sequences were determined by sequencing these vectors. By comparing the sequences to the aptamer primer sequences, PR1 and PR2, the aptamer sequences were identified from the vector. Nucleotide sequences of the aptamers are presented in the Table below.
  • the length of the original DNA library, where the aptamers have been selected from was 100 nucleotide (27 nt and 33 nt long primer binding sites and 40 nt random sequence). In the Table below the 40 nt random sequence is underlined and the primer binding sites are on both sides of the sequences.
  • the T7 and Sp6r sequences of each sample are complement strands to each other as they are sequenced from the same vector in different directions. Some of the aptamer sequences start with the same sequence as the primer PR1 and some of the sequences start with a sequence of primer PR2. This is due the unknown direction when the insert is ligated into the vector.
  • the first sample was 99 nt long (1 CI-AptK12).
  • aptamers For further analysis, only the 100 nt aptamers (2CI-AptK12, 3CI-AptK12 and 4CI-AptK12) were selected. 1 CI-AptK12 was left out from the further analysis because the sequence did not meet the criteria of 100 nt. In order to synthesise the aptamers the number of nucleotides had to be reduced. The aptamers were analysed using UNAFold program that gives aptamers secondary structures most likely to be formed in the binding buffer conditions. The structures are presented in Figure 44 (2CI-AptK12), Figure 45 (3CI-AptK12) and Figure 46 (4CI-AptK12).
  • the binding of the selected aptamers to £ coli K12 was tested.
  • the aptamers (Table above) were synthesised with a FAM-label and incubated (10, 20, 50 and 100 pmol) with £ coli K12 in triplicate. After the washes the fluorescence was measured using a plate reader and the fluorescence values are presented in Figure 47 (this Figure is a duplication of Figure 2).
  • the specificity of the aptamer was tested by incubating a mixture of the aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 (50 pmol) with overnight grown £. coli B and S. aureus.
  • the positive control sample used was an overnight grown £. coli K12 culture.
  • the mixture of the aptamers was used instead of individual aptamers. After the incubation, samples were washed and the fluorescence was measured by the plate reader. The fluorescence values are presented in Figure 49. The results showed that the fluorescence measured for E. coli K12 was significantly higher than E. coli B and S.
  • the samples were visualised under the microscope with a fluorescence and visible light.
  • the microscope images are presented in Figure 50.
  • the fluorescence images for background samples are on the left hand side, the images taken with fluorescence light in the middle and the visible light images on the right hand side.
  • Bright green dots with a dark background can be detected in the positive control sample with E. coli K12. Not as bright and not as many dots can be seen in samples with E. coli B and S. aureus.
  • This result confirms the aptamers 1AptK12, 2AptK12, 4AptK12 and 6AptK12 are specifically binding to E. coli K12 but a little binding to E coli B and S.
  • aureus can be seen. It can be seen on the fluorescence images that the background is darker when more fluorescence is present in the sample (E coli K12) even though the same exposure time was used in all images (200 ms). It can be possible that the microscope is changing the lightning when not much fluorescence is available in the samples.
  • the specificity of the individual four aptamers 1AptK12, 2AptK12, 4Apt 12 and 6AptK12 was tested against E. coli B and S. aureus. E. coli K12 was used as a positive control.
  • the aptamer concentration used in this experiment was 20 pmol because this amount is showed to be enough to see the differences in the fluorescence values.
  • the number or bacterial cells might vary as the bacterial culture is grown overnight and therefore the age of the cultures might vary.
  • the washing steps might affect to the fluorescence readings. Sometimes more bacterial cells are washed off during the washes. The samples were looked under the microscope. Some bright dots were visible on the microscope images as seen in the images presented in Figure 50. The fluorescence images for these samples are not shown here as the results are similar to the results when the mixture of the aptamers was incubated with the bacterial cells in Figure 50.
  • the aptamer pool 9 specific to £. coli K12 was selected and the binding of the aptamer pool 9 has been tested with a fluorescence based detection method.
  • aptamer pool 9 which binds specifically to E. coli K12, was cloned using a commercial cloning kit to a plasmid vector.
  • the cloned inserts were transformed into competent cells where the insert was enriched.
  • the plasmid vectors were extracted and sequenced.
  • the sequenced aptamers were analysed using the aptamers theoretical secondary structures and the AG values. Four aptamer sequences were synthesised with a fluorescent label. The binding and the specificity of these synthesised aptamers were tested. The results show that the selected aptamers bind specifically to E. coli K12.
  • Aptamers were selected to bind live E. coli K12 cells.
  • the binding properties of aptamer pool 9 were analysed and the aptamer sequences were subsequently cloned. The binding of the identified aptamers to E. coli K12 and the specificity were tested.
  • the activity of the specific E. coli K12 aptamer pool 9 was first tested in tap water and the results were used to develop a detection assay that can be used for the detection of bacteria in yoghurt.
  • the aptamers in natural probiotic yoghurt containing Lactobacillus acidophilus and Bifidobacterium ssp. was first tested with the aptamer pool 9. Once the aptamers were cloned they were produced with fluorescent labels and then used for the detection of bacterial cells from yoghurt. 6.2 METHODS
  • Aptamers selected to bind E. coli K12 have been used to detect live bacterial cells in buffer conditions. Tap water was used to see if the aptamers retained their activity in unbuffered conditions and can still be used for bacterial detection. Tap water was spiked with an excess of E. coli K12 bacterial cells. The overnight grown bacterial suspension (1 ml) was centrifuged (2.3.9.1) and the bacterial pellet was resuspended into 1 ml of tap water. The aptamers (10 pmol and 20 pmol) were added into the solution and incubated for 45 min. The control sample without the aptamers was also prepared. Samples were washed twice with BB and the fluorescence was measured by a plate reader. 2 Detection of E. coli K12 from probiotic yoghurt
  • a method was developed to detect bacterial cells from food samples.
  • Natural probiotic yoghurt that contains live cultures of L. acidophilus and Bifidobacterium ssp. was used as the food matrix.
  • Fluorescent FAM-labelled aptamers were produced (2.3.7) and an overnight culture of E. coli K12 (10 ml) was prepared (2.3.9.1) and resuspended into 4 ml of binding buffer (BB).
  • Yoghurt samples were prepared by mixing 3 ml of natural probiotic yoghurt and 3 ml of BB for negative samples and 3 ml of yoghurt, 2 ml of BB and 1 ml of bacterial suspension for the samples.
  • the further optimised detection method was done to detect the E. coli K12 ceils from yoghurt with the aptamers.
  • the sample sizes were reduced and less E. coli K12 bacterial cells added to make it possible to perform this experiment in microcentrifuge tubes.
  • the FAM-labelled aptamers were produced (2.3.7) and the bacterial cells were prepared (2.3.9.1 ).
  • the bacterial suspension (3 ml) was washed and re-suspended into 1500 ⁇ of BB.
  • the yoghurt samples were 750 ⁇ of probiotic yoghurt and 750 ⁇ of BB for the negative control samples.
  • coli K12 spiked yoghurt samples were 750 ⁇ of yoghurt and 500 ⁇ of bacterial suspension topped up with BB to the final volume of 1000 ⁇ .
  • the samples were mixed well and the bacteria were separated from the yoghurt by centrifuging the samples for 5 minutes at 1000g. This centrifugation speed and time was lowered from the time and speed previously used (15000, 10 min) because this way more bacterial cells could be separated from the yoghurt.
  • Supernatant that contained bacterial cells, was collected.
  • an additional 200 ⁇ of BB was added and after samples were mixed they were centrifuged as above. The supernatant was collected and added to the samples.
  • the samples were now centrifuged at 3500a; for 5 minutes and washed twice with BB.
  • the bacterial cells were resuspended into 100 ⁇ of BB with 20 pmol FAM-labelled aptamers.
  • Control samples with no aptamers were done by resuspending the cells into 100 ⁇ BB.
  • the yoghurt samples prepared for analysis are summarised in the Table below.
  • the fluorescence values were read by the plate reader (2.3.9.3) and the results were analysed with the analysis of variance (ANOVA).
  • the aptamer pool 9 was cloned (5.3.1.1 ). Four aptamer sequences (5.3.3) were synthesised and the binding properties of these aptamers were tested (5.3.4).
  • the yoghurt samples were prepared as described above (6.2.2.2). The bacterial cells were extracted from the yoghurt and a 20 pmol aptamer mixture contained 5 pmol of each of the cloned FAM-labelled aptamers was incubated with the samples in triplicate. Samples were incubated for 45 min and after the washes the fluorescence was measured by the plate reader. Difference between the fluorescence values was analysed with the ANOVA.
  • the activity of the FAM-labelled aptamer pool 9 was first tested in tap water that had been spiked with E. coli K12 bacterial cells.
  • the fluorescence intensity values are presented in Figure 52.
  • Two aptamer concentrations were used (10 pmol and 20 pmol) and it can be seen in the graph that the fluorescence detected for 10 pmol sample is almost half of the fluorescence detected for the 20 pmol sample where the aptamer amount was twice as much. This result shows that the aptamers were active in water and that they can be used to detect bacterial cells in water samples. 2 Detection of E. coli K12 from probiotic yoghurt
  • the bacterial cells including the bacteria in yoghurt, were extracted and it might be possible that some components from yoghurt have remained in the samples and the aptamers have bound to them. It is also possible that the unbound aptamers are not washed off properly.
  • One possibility is that some binding to Bifidobacterium ssp. takes place and therefore some fluorescence can be detected.
  • the results presented here show that the fluorescent-labelled aptamers can be used to detect live bacterial cells from yoghurt.
  • Aptamers were used to detect bacterial cells from water and yoghurt. Thus the aptamers can be used in a new type of detection method for food poisoning bacteria.
  • non- pathogenic E. coli K12 was used as an example strain but aptamers can also be used in a detection of pathogens.
  • Aptamers can be used to detect the bacterial cells in different types of food matrices, for example in solid matrices such as meat or cheese. Aptamers could possibly be added on the surface of the meat and the non-binding aptamers could be washed off. The fluorescence can then be measured or visualised.
  • EXAMPLE 7 Selection of the aptamers against pathogenic bacteria
  • Aptamers have been shown herein to be a tool in the development of rapid detection methods for bacteria such as, food-borne pathogens.
  • the selection method for aptamers was developed and the aptamers were selected to bind to non-pathogenic E. coli K12 live bacterial cells.
  • the aptamers were then cloned, sequenced and the binding and the specificity of these selected sequences were tested by using a method based on fluorescence.
  • the aptamer selection method was applied to the food-borne pathogenic bacteria.
  • the aptamers were selected to bind to two different pathogenic E. coli strains including 0157, three Listeria strains (L innocua and two species of L. monocytogenes) and two Salmonella strains (S. typhimurium and S. enteritidis).
  • the aptamers against pathogenic E. coli were selected from an existing pool of E. coli K12 binding aptamers while the selection process that was previously described was used to select the aptamers against Listeria and Salmonella. From these aptamers two pools having the best characteristics were selected for further analysis.
  • three aptamer sequences for E. coli 0157 and three sequences for S. typhimurium were identified and the binding was tested. Some of the sequences showed specific binding and good affinity against their target bacteria.
  • a DNA library was produced as described before (2.2.3) and the aptamers were selected to bind to pathogenic bacterial strains. The selection of the aptamers was done following the protocol (2.3.6) with one exception; the counter selection was not performed. The counter selection was left out in order to see if the counter selection is necessary in terms of the specificity of the aptamers but also for time saving reasons. 7.2.1.1 Aptamers against Escherichia coli 496 and 0157 497
  • Aptamers were selected to bind to two strains of pathogenic E. coli from pool 9 of E. coli K12 binding aptamers. It is possible the aptamer pool 9 binds to some structures on the surface of E. coli K12 that are the same as on the surface of the pathogenic E. coli strains. Selection was done as a normal aptamer selection (2.3.6) except that the selection process was only performed once. The selection and the counter selection were done before as described (3.3.2). After the incubation the samples were washed three times. The aptamers were collected and amplified by PCR (2.3.1 ) and the samples were separated on agarose gel
  • the aptamers were selected from the random DNA library to bind to L. innocua 17, L monocytogenes 489 and L monocytogenes 490 as previously described (2.3.6), except the counter selection was not performed.
  • the aptamers were selected for all three strains until aptamer pool 7. The only successfully selected aptamer pool 7 was for L. monocytogenes 490.
  • This sample was divided in two tubes and the aptamers were further selected in duplicate. To increase the PCR yield the template was added to the reaction in volume 1.5 ⁇ instead of 1 ⁇ .
  • the aptamers were collected and amplified by PCR (2.3.1) and the samples were separated on agarose gel (2.3.2).
  • the aptamers were selected from a random DNA library to bind to S. typhimurium 223 and S. enteritidis 1152 following the selection protocol (2.3.6). The selection without the counter selection steps was repeated nine times. After each selection round the aptamers were collected and amplified by PCR (2.3.1) and the samples were separated on agarose gel (2.3.2). .2 Fluorimetry detection of the pathogen binding aptamer pools
  • the aptamer pools were fluorescent (FAM) labelled (2.3.7) and the binding of the aptamers was tested by the fluorimetry analysis (2.3.9.3).
  • FAM fluorescent
  • Four PCR products were mixed together, purified and used in the binding reaction.
  • the binding was tested for the aptamers that were selected against E. coli 496, E. coli 497, L. monocytogenes 490, S. typhimurium 223 and S. enteritidis 1 152.
  • the binding of the previously selected E. coli K12 aptamers (3.3.2) was tested parallel.
  • the DNA concentration measurement was not used in this study but the fluorescence of the aptamers was measured before they were mixed with the samples. 3 Cloning of the aptamers
  • the ninth aptamer pools against E coli 0157 497 and S. typhimurium 223 were selected for cloning.
  • the pGEM-T Easy vector cloning was done as previously described (2.3.12.1 ) and the same protocol was followed as for the cloning of E. co// K12 aptamers (5.2.1).
  • the aptamer sequences were determined by sequencing the plasmid vectors with inserted aptamers. The colonies that were showed to be positive in PCR analysis were inoculated into LB-media and the plasmid was purified from the overnight grown culture (2.3.12.4). Six plasmid DNA samples (30 ⁇ ), three of each strain, were sequenced (2.3.12.6). The 100 bases long aptamer sequences were identified from the vector sequence.
  • Aptamer secondary structures make the binding of the aptamer to their target possible.
  • the structures were analysed as previously described (2.3.12.7).
  • Three aptamers for E. coli 0157 497 and three aptamers for S. typhimurium 223 were selected and synthesised with a fluorescent FAM label. These aptamers were selected because of their matching structure to the original 100 nucleotides secondary structure.
  • the energy needed to break down the structure ( ⁇ ) that also represents the strength of the structure was taken into account when selecting the aptamers to be synthesised. .5 Binding of the cloned aptamers
  • the aptamers to bind to £ coli 0157 497 were selected from a pool of £ coli K12 binding aptamers. The binding of these aptamers against £ coli K12 was tested by incubating 20 pmol of the aptamer 1Apt497, 2Apt497 and 5AptK12 with £ coli K12 bacterial cells and then measuring the binding by fluorimetry test (2.3.9.3). £ coli K12 specific aptamer 4Apt497 was used as a positive control. The fluorescence was measured with a sensitivity of 75 instead of 50. The fluorescence values were analysed using the statistic test ANOVA. The samples were then visualised and the images were taken under the fluorescence microscope with a 60x magnification (2.3.9.2).
  • the specificity of the S. typhimurium binding aptamers 2Apt223, 3Apt223 and 5Apt223 was tested.
  • the aptamers (20 pmol) were incubated with £ coli 12, L piantarum, S. enteritidis and S. typhimurium 223 (2.3.9.1 ) in triplicate. After the incubation, the samples were washed and the fluorescence was measured by the plate reader (2.3.9.3) and the samples were visualised under the fluorescence microscope (2.3.9.2). The fluorescence values were analysed using the statistic test ANOVA.
  • the aptamers were selected from the random DNA library that has previously been created (3.3.1 ).
  • An agarose gel image of the DNA library can be seen in Figure 15.
  • the bacterial control samples are in lanes 5 and 6. No amplification can be detected in these samples as expected. A faint band can be seen in the DNA control sample in lane 7. This indicates some aptamers that are not necessarily binding to £ coli are remaining in the tube after the washes.
  • Aptamers were selected to bind to three different Listeria strains: L. innocua 17, L. monocytogenes 489 and L. monocytogenes 490. Only seven rounds of selection were repeated for L. innocua 17 and L monocytogenes 489, because no PCR amplification could be seen after the sixth round of selection. For L. monocytogenes 490 nine rounds of selection were performed. After each round the PCR products were separated on agarose gel to see the 100 bp product. The selection of the aptamer pools 1 , 2, 3 and 4 was successful even though the PCR resulted only in faint bands on agarose gels. The gel images are not presented here.
  • Figure 57 is an agarose gel images of the aptamer pools 5, 6 and 7. It can be seen that the aptamers for L innocua 17 in lanes 1 and 2 on gel 7.2a, 7.2c and 7.2d (black boxes) have faint bands. The bands can also be seen in the L monocytogenes 489 aptamers in lanes 1 and 2 on gel 7.2b and lanes 3 and 4 on gel 7.2c and 7.2d (grey boxes). Aptamers for L. monocytogenes 490 in lanes 3 and 4 on gel 7.2b (white boxes) have the strongest bands.
  • aptamer pool 6 and 7 only one of the 490 aptamers appears to have a strong band on the gel (lane 5 on gel 7.2c and 7.2d). This indicates there is more aptamer binding to the bacterial cells in this sample comparing to two other Listeria strains. This strong band was extracted, divided into two and used for further selection.
  • the PCR control, where no aptamer template was added, is in lane 0 on both of the gels 7.2a and 7.2b.
  • the bacterial control and DNA control resulted in clear bands but the results are not seen in the gel images shown in Figure 57.
  • Aptamers were selected to bind to S. typhimurium 223 and S. enteritidis 1 152. Nine rounds of selection were performed. The agarose gels of aptamer pool 1 , 2, 3 and 4 are shown in Figure 59 and pool 5 in Figure 60.
  • the aptamers for S. typhimurium 223 are in lanes 1 and 2 (black box) and the bacterial control sample is in lane 3.
  • the aptamers for S. enteritidis 1152 are in lanes 4 and 5 (white box), and the bacterial control is in lane 6.
  • the DNA control sample where no bacterial cells were added is in lane 7. It can be seen in Figure 59 that the bands for the S.
  • the aptamer pools 6, 7, 8 and 9 are shown in Figure 61 .
  • the aptamers for S. typhimurium 223 are marked with black boxes and for S. enteritidis 1152 are in white boxes.
  • the results show that the aptamer pools for S. typhimurium 223 (black boxes) have stronger bands than the S. enteritidis 1 152 for all the pools (white boxes).
  • the PCR control samples (lanes 0) are clear as expected. Bacterial and DNA control samples were done for all selection rounds but can only be seen on gel 7.6a in lanes 1 -3 and on gel 7.6c in lanes 5-7. No amplification was seen in the bacterial control samples. The DNA control sample on gel 7.6c in lane 7 has a faint band.
  • the aptamer pools were fluorescent (FAM) labelled (2.3.7) and the binding was detected by the fluorimetry analysis. Instead of measuring the DNA concentration of the aptamers, in this study, the fluorescence values of the aptamers were measured before the aptamers were added to the bacterial cells.
  • Aptamer pool 9 was previously selected for E. coli K12 (3.3.2) and in this study aptamers were selected against E. coli 496, E coli 497, L. monocytogenes 490, S. typhimurium 223 and S. enteritidis 1 152 and the PCR products were separated on agarose gels. The binding of the aptamers was tested with the previously developed fluorimetry detection assay (4.2.1.3). The binding of the E. coli K12 aptamers to their target has previously been demonstrated (3.3.4; 4.3.1 ) and this aptamer pool was used as a positive control in this experiment. The fluorescence values of the pathogen samples are shown in the Table below.
  • enteritidis 1 152 aptamers did not yield to a strong PCR product ( Figure 61 ) and therefore the fluorescence is also very small compared to S. typhimurium 223 aptamers.
  • S. typhimurium 223 aptamers also have a strong band on a gel ( Figure 61 c) and the fluorescence is much higher in purified aptamers (see Table below, row 0).
  • the aptamer concentrations could have been optimised to this experiment by producing more aptamers by PCR. This would have required several PCR reactions as in a normal binding reaction, when the aptamer concentration is higher, eight PCR reactions were needed.
  • Two aptamer pools were decided to be chosen for further experiments because of the limited time and limited handling resources.
  • the aptamer pool 9 against E. coli 0157 497 and the aptamer pool 9 against S. typhimurium 223 were shown to have the best binding properties according to the results presented here and were therefore chosen for further
  • the aptamer pool 9 against E. coli 0157 and the aptamer pool 9 against S. typhimurium 223 were shown to have the best binding properties and were therefore chosen for further experiments. These pools of aptamers with unknown sequences were cloned and sequenced to determine the nucleotide sequence. Aptamer pools 9 were amplified by PCR and cloned with pGEM-T Easy vector and JM109 competent cells. The colonies on indicator plates, containing ampicillin, X-Gal and IPTG were counted and a number of positive (white) and negative (blue) colonies is presented in the Table below.
  • the colonies are only growing on the plates due the ampicillin resistance of the bacteria that has been gained by a successful ligation of the insert into the vector.
  • the background is representing the number of bacteria without the vector growing on the plate and the positive control sample is representing a successful transformation into the competent cells.
  • six white colonies were selected and streaked on new selective plates. After an overnight incubation on selective plates all of these colonies that were white on the first plate were expressing the blue colour (CI1s-CI6s, CI1e, CI3e-CI6e), expect one sample that remained white (CI2e). Sometimes the blue colour may be expressed even though the insert has been ligated into the vector, as previously discussed (5.3.1.1 ).
  • the negative control was amplified by using a colony from cloning ligation control. This colony contained a control insert that does not have a binding site for the aptamer primers PR1 and PR2 and therefore was not expected to produce any PCR products. .4 Sequencing of cloned vectors and aptamer analysis
  • the cloned aptamer sequences against pathogenic E. coli 0157 497 (CI2e-CI6e) and S. typ imurium 223 (CI1 s-CI6s) were further analysed in order to obtain the DNA sequence.
  • the positive colonies were transferred into a broth and incubated overnight.
  • the plasmid DNA was purified from the overnight culture and the plasmid quality was tested by separating the samples on an agarose gel.
  • Figure 63 the agarose gel image with the plasmid vectors is shown. It can be seen in the figure that all the other bands are the same size except the band in lane CI2e. This band is larger in size than the other bands and has also a faint band just above the dark band.
  • aptamers In order to synthesise the aptamers the number of nucleotides had to be reduced (James, 2000).
  • the aptamer sequences (rows T7 in the Table above) 3CI-Apt497, 4CI-Apt497, 5CI- Apt497, 1 CI-Apt223, 2CI-Apt223, 3CI-Apt223 and 4CI-Apt223 were further analysed using the UNAFold program that gives aptamer secondary structures most likely to be formed in buffer conditions.
  • aptamer 5Apt497 ( Figure 66) only one structure is shown because this is the only way the sequence can fold according to UNAFold.
  • the ⁇ was calculated by the UNAFold and it is defined as a change in Gibbs energy when the system undergoes a thermodynamic change.
  • two different sequences were isolated from the 100 bp sequence but in some sequences only one sequence could have be isolated (3CI-Apt497, 2CI-Apt223, 4CI-Apt223).
  • the secondary structures as well as energy needed to break the structure (AG) were compared and the three strongest aptamers for each pathogenic strain (S. typhimurium and E. coli 0157) were selected and synthesised with FAM-labels.
  • the synthesised aptamers along with their size and AG values are listed in the Table below.
  • the aptamer 4Apt497 was selected instead of aptamer 5Apt497 that has a AG value -3.08 because aptamer 4Apt497 has only a one secondary structure ( Figure 66). It would be interesting to see if this type of aptamer binds better than the aptamers that can form different type of secondary structures. These six aptamers were further analysed.
  • Aptamers selected to bind to pathogenic E. coli 0157 497 were cloned and sequenced.
  • the aptamer sequences (1Apt497, 2Apt497 and 4Apt497) were synthesised with a fluorescent label and the binding was tested against live E. coli 0157 497 bacterial cells.
  • the aptamers (10, 20 and 50 pmol) were incubated with the bacterial cells in triplicate. The binding was analysed with a fluorimetry analysis and the samples were examined under the fluorescence microscope. The fluorescence values are presented in Figure 71. The result shows that when the aptamers have been added the fluorescence values are greater.
  • the fluorescent FAM label that is attached to the aptamers is only a small molecule (6-Carboxyfluorescein). If only one aptamer has bound to a bacterial cell it is very likely that this bacterial cell is not visible in the microscope image. Although some bright dots that were not visible in background samples can be detected in the image where the aptamers were added. This result indicates that some aptamer binding can be detected. E. coli 0157 bacterial cells are relatively small in size and move rapidly on the microscope slide. This made the visualisation of the bacterial cells challenging and therefore some of the images are not clear. Bright green bacterial cells were often seen when two bacterial cells were connected to each other (dividing cells). This can be seen for example in the fluorescence images of sample 2Apt497 and 4Apt497.
  • £. coli 0157 aptamers were selected from a pool of £. coli K12 binding aptamers.
  • the binding of these three cloned aptamers 1Apt497, 2Apt497 and 4Apt497 was tested against E. coli K12 by the fluorimetry analysis.
  • the aptamers (20 pmol) were incubated with the bacterial cells and the fluorescence was measured.
  • Aptamer 4AptK12 was used as a positive control as the binding of this aptamer has previously been demonstrated (5.3.4.1).
  • the fluorescence values that are presented in Figure 73 were measured for all the samples and some binding can be detected from each sample.
  • the fluorescence images are on left hand side and the visible light images on right hand side. It can be seen in the images that when the £ coli K12 bacteria cells were incubated with the aptamer 4AptK12 (positive control), bright green dots can be seen. Some fluorescent dots can also be seen when E. coli K12 was incubated with the pathogen aptamers 1Apt497, 2Apt497 and 4Apt497 but the fluorescence is not as bright in the aptamer 4AptK12 image. This result confirms some binding of the aptamer 1Apt497, 2Apt497 and 4Apt497 to E. coli K12 can be detected.
  • the aptamers that were selected to bind to S. typhimurium 223 were cloned and the sequences were synthesised with fluorescent labels.
  • the binding of the FAM-aptamer sequences 2Apt223, 3Apt223 and 5Apt223 was tested against live S. typhimurium 223 bacterial cells.
  • the aptamers (10, 20 and 50 pmol) were incubated with the bacterial cells in triplicate. The binding was analysed with the fluorimetry analysis. The fluorescence values are presented in Figure 75. The results show that the greater the number of added aptamers the higher the fluorescence.
  • the results indicate that the addition of 10 pmol of S.
  • the samples were visualised under a microscope with a green fluorescent light and a visible light. There was no great difference between the images taken of the samples with different aptamer concentrations.
  • the images for 20 pmol are shown in Figure 76.
  • the fluorescence microscope images are on the left hand side and the visible light images are on the right hand side.
  • the bacterial cells with fluorescent labelled aptamers bound to them can be seen on the images as bright green dots. No aptamers were added to the background samples. In the images not many bright green bacterial cells can be seen. It is possible that the aptamers are only binding to some of the bacterial cell or only a small number of aptamers is binding to cell and therefore the bacterial cells cannot be detected in the images.
  • the specificity of the S. typhimurium aptamers 2Apt223, 3Apt223 and 5Apt223 was tested.
  • the aptamers (20 pmol) were incubated with E. coli K12, L. plantarum and S. enteritidis and S. typhimurium.
  • the samples were visualised under the microscope.
  • the microscope images for S. typhimurium, S. enteritidis, E. coli K12 and L plantarum samples with 3Apt223 aptamers are presented in Figure 78 and Figure 79.
  • the visible light images are presented on the left hand side and the fluorescence images on the right hand side.
  • Samples 2Apt223 and 5Apt223 are not presented here because there is no great difference between the samples.
  • a bright spot can be seen in the S. typhimurium fluorescence image (left hand side Figure 78) when the aptamers have been added.
  • S. typhimurium fluorescence image left hand side Figure 78
  • Aptamers were selected to bind to pathogenic E. coli 496, E. coli 0 57 497, L. innocua 17, L monocytogenes 489, L monocytogenes 490, S. enteritidis 1 152 and S. typhimurium 223.
  • the binding of the aptamer pools was tested against their targets.
  • the ninth aptamer pools against E. coli 0157 497 and S. typhimurium 223 were selected for cloning and sequencing.
  • the aptamer structures were then analysed and three sequences for each strain were synthesised with fluorescent labels.
  • the binding was detected when the samples were separated on the agarose gel.
  • Two aptamer pools against pathogens were then selected for cloning and sequencing. The binding of these aptamers was then tested against their targets.
  • the three synthesised sequences against pathogenic E. coli 0157 were showing some binding and the aptamers against S. typhimurium showed a good binding at the fluorimetry analysis. Some differences in binding were detected between different aptamers.
  • aptamers can also bind to E. coli K12 but the binding is not as strong as the binding of the E. coli K12 specific aptamers.
  • the specificity test that was performed to S. typhimurium aptamers showed that some of the aptamers were capable of detecting other bacterial cells and only some of the aptamers were specific to S. typhimurium. This study showed that the aptamers can be selected to bind to food-borne pathogens.
  • aptamers can be easily selected to specifically bind to live bacterial cells. Unlike the usual aptamer selection process where aptamers are selected to bind to extracted surface molecules, the inventors show herein that aptamers are successfully selected to bind to whole bacterial cells. Most importantly the inventors have also shown that these aptamers can be used in the detection of pathogens (e.g. food-borne pathogens) in complex matrixes, e.g. in contaminated food.
  • pathogens e.g. food-borne pathogens
  • RNA aptamers selected to bind Human Immunodeficiency Virus Type 1 Rev in vitro are:

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Abstract

La présente invention concerne un aptamère d'acide nucléique comprenant la séquence nucléotide présentée ici en tant que SEQ ID No. 5, 6, 7, 8, 9, 10, 1, 2, 3 ou 4 ou son fragment, ou une séquence qui lui est identique à au moins 80 %. L'invention porte en outre sur l'utilisation d'aptamères nucléiques pour détecter la présence de bactéries pathogènes dans un échantillon, en particulier dans une matrice complexe, telle qu'un système alimentaire.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105238852A (zh) * 2015-08-10 2016-01-13 济南大学 基于核酸适配体检测鼠伤寒沙门氏菌的生物传感器及其制备方法
CN106929510A (zh) * 2016-12-23 2017-07-07 中国人民解放军军事医学科学院卫生学环境医学研究所 一种特异性核酸适配体及应用
US10526663B2 (en) 2014-02-21 2020-01-07 Merck Patent Gmbh Method of detecting a microorganism in a sample by a fluorescence based detection method
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WO2023287511A3 (fr) * 2021-06-02 2024-01-11 Board Of Regents, The University Of Texas System Procédés et compositions associés à des biocapteurs transformés

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9891227B2 (en) * 2014-05-02 2018-02-13 Academia Sinica Ultrasensitive detection of a biological target by aptamer-conjugated gold nanoparticles
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6569630B1 (en) 1999-07-02 2003-05-27 Conceptual Mindworks, Inc. Methods and compositions for aptamers against anthrax
US20090004644A1 (en) * 2006-12-28 2009-01-01 Conceptual Mindworks, Inc Methods and compositions for processes of rapid selection and production of nucleic acid aptamers
WO2009070749A1 (fr) * 2007-11-30 2009-06-04 Regents Of The University Of Minnesota Aptamères d'adn
US20090186342A1 (en) * 2006-05-12 2009-07-23 Pronucleotein Biotechnologies, Llc Methods of producing competitive aptamer fret reagents and assays
WO2009104075A2 (fr) * 2008-02-21 2009-08-27 Otc Biotechnologies, Llc Procédés de production de conjugué fluorophore-bille magnétique-aptamère adhérent au plastique et autres dosages sandwich
WO2011097420A2 (fr) * 2010-02-03 2011-08-11 North Carolina State University Sélection et caractérisation d'aptamères d'adn dotés d'une sélectivité de liaison à campylobacter jejuni en utilisant le procédé selex des cellules entières

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6569630B1 (en) 1999-07-02 2003-05-27 Conceptual Mindworks, Inc. Methods and compositions for aptamers against anthrax
US20090186342A1 (en) * 2006-05-12 2009-07-23 Pronucleotein Biotechnologies, Llc Methods of producing competitive aptamer fret reagents and assays
US20090004644A1 (en) * 2006-12-28 2009-01-01 Conceptual Mindworks, Inc Methods and compositions for processes of rapid selection and production of nucleic acid aptamers
WO2009070749A1 (fr) * 2007-11-30 2009-06-04 Regents Of The University Of Minnesota Aptamères d'adn
WO2009104075A2 (fr) * 2008-02-21 2009-08-27 Otc Biotechnologies, Llc Procédés de production de conjugué fluorophore-bille magnétique-aptamère adhérent au plastique et autres dosages sandwich
WO2011097420A2 (fr) * 2010-02-03 2011-08-11 North Carolina State University Sélection et caractérisation d'aptamères d'adn dotés d'une sélectivité de liaison à campylobacter jejuni en utilisant le procédé selex des cellules entières

Non-Patent Citations (78)

* Cited by examiner, † Cited by third party
Title
A EUROPEAN JOURNAL, vol. 10, 2004, pages 1868 - 1876
ALTSCHUL ET AL., J. MOL. BIOL., 1990, pages 403 - 410
ANURAG JYOTI ET AL: "Identification of Environmental Reservoirs of Nontyphoidal Salmonellosis: Aptamer-Assisted Bioconcentration and Subsequent Detection of Salmonella Typhimurium by Quantitative Polymerase Chain Reaction", ENVIRONMENTAL SCIENCE & TECHNOLOGY, vol. 45, no. 20, 15 October 2011 (2011-10-15), pages 8996 - 9002, XP055030651, ISSN: 0013-936X, DOI: 10.1021/es2018994 *
BAEUMNER ET AL., BIOSENSORS AND BIOELECTRONICS, vol. 18, 2003, pages 405 - 413
BALDRICH ET AL., ANALYTICAL CHEMISTRY, vol. 76, 2004, pages 7053 - 7063
BONWICK, G.A.; SMITH, C.J.: "Immunoassays: their history, development and current place in food science and technology", INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY, vol. 39, 2004, pages 817 - 827
BRITTANY BOOK ET AL: "Quantification of receptor targeting aptamer binding characteristics using single-molecule spectroscopy", BIOTECHNOLOGY AND BIOENGINEERING, vol. 108, no. 5, 1 May 2011 (2011-05-01), pages 1222 - 1227, XP055056144, ISSN: 0006-3592, DOI: 10.1002/bit.23043 *
CAMILLE L A HAMULA ET AL: "Selection and analytical applications of aptamers binding microbial pathogens", TRAC, TRENDS IN ANALYTICAL CHEMISTRY, vol. 30, no. 10, 9 September 2011 (2011-09-09), pages 1587 - 1597, XP028310101, ISSN: 0165-9936, [retrieved on 20110910], DOI: 10.1016/J.TRAC.2011.08.006 *
CAMILLE L. A. HAMULA ET AL: "Selection of Aptamers against Live Bacterial Cells", ANALYTICAL CHEMISTRY, vol. 80, no. 20, 15 October 2008 (2008-10-15), pages 7812 - 7819, XP055030661, ISSN: 0003-2700, DOI: 10.1021/ac801272s *
CAMILLE L.A. HAMULA ET AL: "DNA Aptamers Binding to Multiple Prevalent M-Types of Streptococcus pyogenes", ANALYTICAL CHEMISTRY, vol. 83, no. 10, 15 May 2011 (2011-05-15), pages 3640 - 3647, XP055030659, ISSN: 0003-2700, DOI: 10.1021/ac200575e *
CAO, X.; LI, S.; CHEN, L.; DING, H.; XU, H.; HUANG, Y.; LI, J.; LIU, N.; CAO W.; ZHU, Y.: "Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus", NUCLEIC ACIDS RESEARCH, vol. 37, 2009, pages 4621 - 4628
CARUTHERS MH ET AL., NUC ACIDS RES SYMP SER, 1980, pages 215 - 23
CHEN ET AL: "Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 357, no. 3, 27 April 2007 (2007-04-27), pages 743 - 748, XP022138305, ISSN: 0006-291X, DOI: 10.1016/J.BBRC.2007.04.007 *
CHENG ET AL., BIOELECTROCHEMISTRY, vol. 77, 2009, pages 1 - 12
CHENG, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 357, 2007, pages 743 - 748
ELLINGTON, A.D.; SZOSTAK J.W.: "In vitro selection of RNA molecules that bind specific ligands", NATURE, vol. 346, 1990, pages 818 - 822
FEMS MICROBIOL LETT, vol. 174, no. 2, 1999, pages 247 - 50
FEMS MICROBIOL LETT, vol. 177, no. 1, 1999, pages 187 - 8
FREEMAN ET AL., ANALYTICAL CHEMISTRY, vol. 84, 2012, pages 6192 - 9198
GUSTAVO A. ZELADA-GUILLÉN ET AL: "Real-Time Potentiometric Detection of Bacteria in Complex Samples", ANALYTICAL CHEMISTRY, vol. 82, no. 22, 15 November 2010 (2010-11-15), pages 9254 - 9260, XP055050457, ISSN: 0003-2700, DOI: 10.1021/ac101739b *
HALE; MARHAM: "THE HARPER COLLINS DICTIONARY OF BIOLOGY", 1991, HARPER PERENNIAL
HALL ET AL., BIOTECHNOLOGY AND BIOENGINEERING, vol. 103, 2009, pages 1049 - 1059
HAMULA ET AL., TRENDS IN ANALYTICAL CHEMISTRY, vol. 25, 2008, pages 681 - 691
HAMULA, C.L.A.; ZHANG, H.; GUAN, L.L.; LI, X-F.; LE, X.C.: "Selection of aptamers against live bacterial cells", ANALYTICAL CHEMISTRY, vol. 80, 2008, pages 7812 - 7819
HARI P DWIVEDI ET AL: "Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 87, no. 6, 27 June 2010 (2010-06-27), pages 2323 - 2334, XP019841726, ISSN: 1432-0614 *
HERNE ET AL., JOURNAL OF AMERICAN CHEMISTRY SOCIATY, vol. 119, 1997, pages 8916 - 8920
HIANIK ET AL., BIOELECTROCHEMISTRY, vol. 70, 2007, pages 127 - 133
HIGGINS DG; SHARP PM, GENE, vol. 73, no. 1, 1988, pages 237 - 244
HO ET AL., ANALYTICAL CHEMISTRY, vol. 84, 2012, pages 4245 - 4247
HORN T ET AL., NUC ACIDS RES SYMP SER, 1980, pages 225 - 232
IQBAL, S.S.; MAYO, M.W.; BRUNO, J.G.; BRONK, B.V.; BATT, C.A.; CHAMBERS, J.P: "A review of molecular recognition technologies for detection of biological threat agents", BIOSENSORS & BIOELECTRONICS, vol. 15, 2000, pages 549 - 578
J. G. BRUNO ET AL: "In Vitro antibacterial effects of antilipopolysaccharide DNA aptamer-C1qrs complexes", FOLIA MICROBIOLOGICA, vol. 53, no. 4, 1 July 2008 (2008-07-01), pages 295 - 302, XP055030719, ISSN: 0015-5632, DOI: 10.1007/s12223-008-0046-6 *
JOSHI R ET AL: "Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars", MOLECULAR AND CELLULAR PROBES, ACADEMIC PRESS, LONDON, GB, vol. 23, no. 1, 1 February 2009 (2009-02-01), pages 20 - 28, XP025910706, ISSN: 0890-8508, [retrieved on 20081118], DOI: 10.1016/J.MCP.2008.10.006 *
JOSHI, MOLECULAR AND CELLULAR PROBES, vol. 23, 2009, pages 20 - 28
JOSHI, R.; JANAGAMA, H.; DWIVEDI, H.P.; KUMAR T.M.A.S.; JAYKUS, L-A; SCHEFERS, J.; SREEVATSAN, S.: "Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars", MOLECULAR AND CELLULAR PROBES, vol. 23, 2009, pages 20 - 28
KARKKAINEN ET AL., INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY, vol. 46, no. 3, 2011, pages 445 - 454
KARKKAINEN, R.; DRASBEK, M.R.; MCDOWELL, I.; SMITH, C.J.; YOUNG, N.W.G.; BONWICK,G.: "Aptamers for safety and quality assurance in the food industry: detection of pathogens", INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY, 2011
KAROONUTHAISIRI, N.; CHARLERMROJ, R.; UAWISETWATHANA, U.; LUXANANIL, P.; KIRTIKARA, K.; GAJANANDANA, O.: "Development of antibody array for simultaneous detection of foodborne pathogens", BIOSENSORS & BIOELECTRONICS, vol. 24, 2009, pages 1641 - 1648
KIM ET AL., ANALYTICAL CHEMISTRY, vol. 84, 2012, pages 6192 - 9198
LAUTNER ET AL., THE ANALYST, vol. 135, 2010, pages 918 - 9
LAUTNER ET AL., THE ANALYST, vol. 135, 2010, pages 918 - 926
LEE ET AL., ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 390, 2008, pages 1023 - 1032
LEE; WALT, ANALYTICAL BIOCHEMISTRY, vol. 282, 2000, pages 142 - 146
LISS ET AL., ANALYTICAL CHEMISTRY, vol. 74, 2002, pages 4488 - 4495
LIU ET AL., ELECTROCHIMICA ACTA, vol. 54, 2009, pages 6207 - 6211
LIU ET AL., ELECTROCHIMICA ACTA, vol. 54, 2010, pages 6207 - 6211
LIU; LU, ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 45, 2006, pages 90 - 94
MCCAULEY ET AL., ANALYTICAL BIOCHEMSITRY, vol. 319, 2003, pages 244 - 250
MIN ET AL., BIOSENSORS AND BIOELECTRONICS, vol. 23, 2008, pages 1819 - 1824
MINUNNI ET AL., BIOSENSORS AND BIOELECTRONICS, vol. 20, 2004, pages 1149 - 1156
MUHAMMED- TAHIR; ALOCILJA, BIOSENSORS AND BIOELECTRONICS, vol. 18, 2003, pages 813 - 819
NASIUND ET AL., NATURE METHODS APPLICATION NOTES, 2006, pages 14 - 16
NUTIU; LI, JOURNAL OFAMERICAN CHEMICAL SOCIETY, vol. 125, 2003, pages 4771 - 4778
NUTIU; LI, METHODS, vol. 37, 2005, pages 16 - 25
OHK ET AL., JOURNAL OF APPLIED MICROBIOLOGY, vol. 109, 2010, pages 808 - 817
PENDERGRAST, P.S.; MARSH, H.N.; GRATE, D.; HEALY J.M.; STANTON, M.: "Nucleic acid aptamers for target validation and therapeutic applications", JOURNAL OF BIOMOLECULAR TECHNIQUES, vol. 16, 2005, pages 224 - 234
POTYRAILO ET AL., ANALYTICAL CHEMISTRY, vol. 70, 1998, pages 3419 - 3425
S.H. OHK ET AL: "Antibody-aptamer functionalized fibre-optic biosensor for specific detection of Listeria monocytogenes from food", JOURNAL OF APPLIED MICROBIOLOGY, vol. 109, no. 3, 1 September 2010 (2010-09-01), pages 808 - 817, XP055050735, ISSN: 1364-5072, DOI: 10.1111/j.1365-2672.2010.04709.x *
SANDEEP P RAVINDRANATH ET AL: "SERS driven cross-platform based multiplex pathogen detection", SENSORS AND ACTUATORS B: CHEMICAL: INTERNATIONAL JOURNAL DEVOTED TO RESEARCH AND DEVELOPMENT OF PHYSICAL AND CHEMICAL TRANSDUCERS, ELSEVIER S.A, SWITZERLAND, vol. 152, no. 2, 5 December 2010 (2010-12-05), pages 183 - 190, XP028169930, ISSN: 0925-4005, [retrieved on 20101216], DOI: 10.1016/J.SNB.2010.12.005 *
SASSOLAS ET AL., ELECTROANALYSIS, vol. 21, 2009, pages 1237 - 1250
SINGLETON ET AL.: "DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED.", 1994, JOHN WILEY AND SONS
STEEL ET AL., ANALYTICAL CHEMISTRY, vol. 70, 1998, pages 4670 - 4677
SYMENSMA, T.L.; GIVER, L.; ZAPP, M.; TAKLE, G.B.; ELLINGTON, A.D.: "RNA aptamers selected to bind Human Immunodeficiency Virus Type 1 Rev in vitro are Rev responsive in vivo", JOURNAL OF VIROLOGY, vol. 70, 1996, pages 179 - 187
TAKEMURA, K.; WANG, P.; VORBERG, I.; SUREWICZ, W.; PRIOLA, S.A.; KANTHASAMY, A.; POTTATHIL, R.; CHEN, S.G.; SREEVATSAN, S.: "DNA aptamers that bind to PrPc and not PrPSc show sequence and structure specificity", EXPERIMENTAL BIOLOGY AND MEDICINE, vol. 231, 2006, pages 204 - 214
TOMBELLI ET AL., BIOSENSORS AND BIOELECTRONICS, vol. 20, 2005, pages 2424 - 2434
TOMBELLI, BIOSENSORS AND BIOELECTRONICS, vol. 20, 2005, pages 2424 - 2434
TOMBELLI, S.; MINUNNI, M.; MASCINI, M.: "Aptamers-based assays for diagnostics, environmental and food analysis", BIOMOLECULAR ENGINEERING, vol. 24, 2007, pages 191 - 200
TORRES-CHAVOLLA E ET AL: "Aptasensors for detection of microbial and viral pathogens", BIOSENSORS AND BIOELECTRONICS, ELSEVIER BV, NL, vol. 24, no. 11, 15 July 2009 (2009-07-15), pages 3175 - 3182, XP026150124, ISSN: 0956-5663, [retrieved on 20081125], DOI: 10.1016/J.BIOS.2008.11.010 *
TORRES-CHAVOLLA; ALOCILJA, BIOSENSORS AND BIOELECTRONICS, vol. 24, 2009, pages 3175 - 3182
TUERK, C.; GOLD, L.: "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase", SCIENCE, vol. 249, 1990, pages 505 - 510
TULEUOVA ET AL., ANALYTICAL CHEMISTRY, vol. 82, 2010, pages 1851 - 1857
TYAGI; KRAMER, NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 303 - 308
VIVEKANANDA, J.; KIEL, J.L.: "Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-linked immobilised Sorbent assay", LABORATORY INVESTIGATION, vol. 86, 2006, pages 610 - 618
WANG ET AL., ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 389, 2007, pages 819 - 825
WILLNER; ZAYATS, ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 46, 2007, pages 6408 - 6418
XU ET AL., ANALYTICAL CHEMISTRY, vol. 77, 2005, pages 5107 - 5113
YAMAMOTO ET AL., GENES CELLS, vol. 5, 2000, pages 371 - 388
ZHANG ET AL., SMALL, vol. 4, 2008, pages 1196 - 1200

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